Epiphytic algal flora associated with habitat forming brown seaweed in a central Mediterranean coastal area (Conero Riviera, Adriatic Sea): diversity and relationship with environmental variables

The Cystoseira s.l. complex includes the most common macrophytes in shallow sublittoral habitats of Mediterranean rocky shores, where they form marine forests providing valuable ecosystem services. These communities are considered to be repositories of great marine biodiversity, but the epiphytic assemblages associated with them are still not fully known. In particular, extremely limited information is available about their microalgal epiphytes. We examined the microepiphytic and macroepiphytic communities associated with the brown alga Gongolaria barbata in a coastal area of the central Adriatic shore (Conero Riviera, Italy). We recorded 119 microalgal taxa and 60 macroalgal taxa growing as epiphytes on this species. Diatoms were the dominant microepiphytes, accounting for 83% of the total number of taxa, followed by Dinophyceae (9%), unidentified phytoflagellates (4%) and Cyanobacteria (3%). A slight negative and positive correlation was observed between phytoflagellate abundance and water temperature and DIN (Dissolved Inorganic Nitrogen). Red algae represented the majority of the macroepiphytes recorded, accounting for 60% of the total. Most of the macroalgal epiphytes were ephemeral species with filamentous morphology, and the non-indigenous red alga Melanothamnus japonicus was among the most common epiphytes. Our results confirm the role of the Cystoseira s.l. species as a major repository of marine biodiversity in the Mediterranean. However, they also suggest that our understanding of the diversity of their associated communities is probably still largely incomplete. New studies combining traditional taxonomy and DNA metabarcoding will be essential to advance our knowledge of them

Effects of natural and anthropogenic stressors on fucalean brown seaweeds across different spatial scales in the Mediterranean Sea

Algal habitat-forming forests composed of fucalean brown seaweeds (Cystoseira, Ericaria, and Gongolaria) have severely declined along the Mediterranean coasts, endangering the maintenance of essential ecosystem services. Numerous factors determine the loss of these assemblages and operate at different spatial scales, which must be identified to plan conservation and restoration actions. To explore the critical stressors (natural and anthropogenic) that may cause habitat degradation, we investigated (a) the patterns of variability of fucalean forests in percentage cover (abundance) at three spatial scales (location, forest, transect) by visual estimates and or photographic sampling to identify relevant spatial scales of variation, (b) the correlation between semi-quantitative anthropogenic stressors, individually or cumulatively (MA-LUSI index), including natural stressors (confinement, sea urchin grazing), and percentage cover of functional groups (perennial, semi-perennial) at forest spatial scale. The results showed that impacts from mariculture and urbanization seem to be the main stressors affecting habitat-forming species. In particular, while mariculture, urbanization, and cumulative anthropogenic stress negatively correlated with the percentage cover of perennial fucalean species, the same stressors were positively correlated with the percentage cover of the semi- erennial Cystoseira compressa and C. compressa subsp. pustulata. Our results indicate that human impacts can determine spatial patterns in these fragmented and heterogeneous marine habitats, thus stressing the need of carefully considering scale-dependent ecological processes to support conservation and restoration

Effects of Natural and Anthropogenic Stressors on Fucalean Brown Seaweeds Across Different Spatial Scales in the Mediterranean Sea

Algal habitat-forming forests composed of fucalean brown seaweeds (Cystoseira, Ericaria, and Gongolaria) have severely declined along the Mediterranean coasts, endangering the maintenance of essential ecosystem services. Numerous factors determine the loss of these assemblages and operate at different spatial scales, which must be identified to plan conservation and restoration actions. To explore the critical stressors (natural and anthropogenic) that may cause habitat degradation, we investigated (a) the patterns of variability of fucalean forests in percentage cover (abundance) at three spatial scales (location, forest, transect) by visual estimates and or photographic sampling to identify relevant spatial scales of variation, (b) the correlation between semi-quantitative anthropogenic stressors, individually or cumulatively (MA-LUSI index), including natural stressors (confinement, sea urchin grazing), and percentage cover of functional groups (perennial, semi-perennial) at forest spatial scale. The results showed that impacts from mariculture and urbanization seem to be the main stressors affecting habitat-forming species. In particular, while mariculture, urbanization, and cumulative anthropogenic stress negatively correlated with the percentage cover of perennial fucalean species, the same stressors were positively correlated with the percentage cover of the semi-perennial Cystoseira compressa and C. compressa subsp. pustulata. Our results indicate that human impacts can determine spatial patterns in these fragmented and heterogeneous marine habitats, thus stressing the need of carefully considering scale-dependent ecological processes to support conservation and restoration

Effects of Natural and Anthropogenic Stressors on Fucalean Brown Seaweeds Across Different Spatial Scales in the Mediterranean Sea

Este artículo contiene 14 páginas, 8 figuras, 3 tablas.Algal habitat-forming forests composed of fucalean brown seaweeds (Cystoseira, Ericaria, and Gongolaria) have severely declined along the Mediterranean coasts, endangering the maintenance of essential ecosystem services. Numerous factors determine the loss of these assemblages and operate at different spatial scales, which must be identified to plan conservation and restoration actions. To explore the critical stressors (natural and anthropogenic) that may cause habitat degradation, we investigated (a) the patterns of variability of fucalean forests in percentage cover (abundance) at three spatial scales (location, forest, transect) by visual estimates and or photographic sampling to identify relevant spatial scales of variation, (b) the correlation between semi-quantitative anthropogenic stressors, individually or cumulatively (MA-LUSI index), including natural stressors (confinement, sea urchin grazing), and percentage cover of functional groups (perennial, semi-perennial) at forest spatial scale. The results showed that impacts from mariculture and urbanization seem to be the main stressors affecting habitat-forming species. In particular, while mariculture, urbanization, and cumulative anthropogenic stress negatively correlated with the percentage cover of perennial fucalean species, the same stressors were positively correlated with the percentage cover of the semi-perennial Cystoseira compressa and C. compressa subsp. pustulata. Our results indicate that human impacts can determine spatial patterns in these fragmented and heterogeneous marine habitats, thus stressing the need of carefully considering scale-dependent ecological processes to support conservation and restoration.This study was supported by the European Union’s EASME (Executive Agency for Small and Medium Enterprise) and EMFF (European Maritime and Fisheries fund) as part of the project AFRIMED, “Algal Forest Restoration in the Mediterranean Sea” (under grant agreement no. 789059), http:// afrimed-project.eu/.Peer reviewe

Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and –c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis

HIGH BIOMAS BLOOMS CAUSING FISH KILLS AND OTHER ENVIRONMENTAL IMPACTS

Harmful algal blooms (HABs) have significant impacts on food safety and security through contamination or mass mortalities of aquatic organisms. Indeed, if not properly controlled, aquatic products contaminated with HAB biotoxins are responsible for potentially deadly foodborne diseases and when rapidly growing, HAB consequences include reduced dissolved oxygen in the ocean, dead zones, and mass mortalities of aquatic organisms. Improving HAB forecasting is an opportunity to develop early warning systems for HAB events such as food contamination, mass mortalities, or foodborne diseases. Surveillance systems have been developed to monitor HABs in many countries; however, the lead-time or the type of data (i.e. identification at the Species-level, determination of toxicity) may not be sufficient to take effective action for food safety management measures or other reasons, such as transfer of aquaculture products to other areas. Having early warning systems could help mitigate the impact of HABs and reduce the occurrence of HAB events. In this regard, FAO took the lead in the development of a Joint FAO-IOC-IAEA Technical Guidance for the Implementation of Early Warning Systems for HABs. The document will guide competent authorities and relevant institutions involved in consumer protection or environmental monitoring to implement early warning systems for HABs present in their areas (marine and brackish waters), specifically for those affecting food safety or food security (benthic HABs, fish-killing HABs, pelagic toxic HABs, and cyanobacteria HABs)

Quali-quantitative determination and structural characterization of microalgal toxins in environmental matrices

“Harmful Algal Blooms” (HABs) are massive proliferations of marine microalgae producing biotoxins. These natural phenomena, occurring under particular climatic and environmental conditions, represent a worldwide problem since they pose serious threats to human health and heavily affect economy of coastal areas. Humans may be affected by marine biotoxins through three main exposure routes: the oral route through consumption of contaminated seafood, the respiratory route through inhalation of aerosolized toxins, and the dermal route through direct contact with microalgae and/or contaminated seawater. However, for some marine biotoxins the mechanism through which they exert their toxicity has not been clarified yet. The Ostreopsis phenomenon currently represents the major HAB-related threat to humans in the Mediterranean area. Since the late 1990s, blooms of Ostreopsis cf. ovata have been repetitively recorded all along the Italian coastlines as well as along the Mediterranean coasts of Spain, France, Croatia, Greece and North Africa. Concomitantly, a severe respiratory syndrome has been observed in humans requiring in some cases hospitalization. Previous studies have demonstrated that O. cf. ovata produces congeners of palytoxin (PLTX), (ovatoxins and an isobaric PLTX), one of the most potent marine toxins so far known, originally isolated from soft corals belonging to the genus Palythoa. However, inhalatory toxicity of PLTX and its congeners was completely unknown and the mechanism through which they harm humans through inhalation is still matter of speculation. The increasing spread of the Ostreopsis phenomenon and the ever-growing number of ovatoxins (OVTXs) being discovered makes the need of evaluating their toxicity urgent. The availability of sufficient amounts of well characterized reference material is the cornerstone for the achievement of toxicity data. Beside PLTX congeners, a wide array of marine biotoxins present serious concern for humans. As a result, National and Local authorities in charge of safeguarding public health have enacted monitoring and risk management programs of toxin with the purpose of limiting adverse impacts of HABs; these regulations require routine monitoring of shellfish for toxins and the analysis of water samples for the presence of toxin-producing microalgae. However, the analysis of shellfish and the analysis of microalgae presents number of technical and practical limitations; for these reasons, in recent years, passive sampling (Solid Phase Adsorption Toxins Tracking, SPATT) has been investigated as an alternative mean to highlight the presence of toxic microalgae directly in seawater. In this frame, the aim of this thesis was: • to investigate the mechanism through which O. cf. ovata and Palythoa spp. and the toxins they produce exert their toxicity; • to determine toxin profile and content of Ostreopsis spp. strains from South-East Mediterranean Sea (Cyprus Island and Lebanon), New Zealand and Australia to serve the double purpose of evaluating the risk they present to humans and investigating the presence of novel PLTX analogues by LC-HRMSn; • to use SPATT methods for detection of marine biotoxins; • to perform preliminary studies toward isolation of PLTX congeners. Chapter 1 presents a general introduction. In Chapter 2 the potentially active role of the mucilaginous matrix produced by O. cf. ovata, has been for the first time investigated. In order to better elucidate toxicity dependence on direct/indirect contact, the role of the mucilaginous matrix and the potential differences in toxicity along the growth curve of O. cf. ovata, a toxic bioassay during exponential, stationary and late stationary phases was carried out. Simultaneously, a molecular assay was performed to quantify intact cells or to exclude the presence of the cells. Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) analyses were also carried out to evaluate toxin profile and content in the different treatments. As a result, a higher mortality of model organism (Artemia salina nauplii) was also observed, especially during the late stationary phase, when direct contact between the model organism and intact microalgal cells occurred (LC50-48h < 4 cells/ml on A. salina). Also growth medium devoid of microalgal cells but containing O. cf. ovata mucilage caused significant toxic effects. This finding was also supported by chemical analyses which showed the highest toxin content in pellet extract (95%) and around 5% of toxins in the growth medium holding mucous, while the growth medium devoid of both cells and mucilage did not contain any detectable toxins. Additionally, the connection between mucilaginous matrix and the thecal plates, pores and trychocysts was explored by way of atomic force microscopy (AFM) to investigate the cell surface at a sub-nanometer resolution, providing a pioneering description of cellular features. In Chapter 3 LC-HRMS analysis of both soft coral and seawater from a home marine aquarium involved in a whole family poisoning is reported. Several anecdotal reports exist of aquarium hobbyists that experienced severe respiratory distress and/or skin injury following cleaning operation of home aquaria containing Palythoa sp. soft corals as well as hundreds of cases of respiratory illness and/or dermatitis have been recorded in proximity to the sea concomitantly with algal blooms of Ostreopsis spp. in the Mediterranean area. Both Palythoa spp. and Ostreopsis spp. contain congeners of PLTX whose inhalation hazard is however unknown. In this study, we demonstrate the presence of high levels of PLTXs (PLTX and hydroxy-PLTX) in both soft coral and seawater collected in the marine aquarium. Due to the high toxin levels found in seawater, a procedure for a rapid and efficient determination of palytoxin in seawater was developed. A comparison of symptoms of Palythoa- and Ostreopsis-related inhalatory poisonings showed many similarities including fever, respiratory distress, nausea, and flu-like symptoms. From the chemical and symptomatological data reported herein it is reasonable to hold PLTXs responsible for respiratory disorders following inhalation. Although the exact mechanism through which PLTX congeners exert their inhalatory toxicity is still unknown, this represents a step toward demonstrating that they exert toxic effects through inhalation both in natural environments and in the surroundings of private and public aquaria. In Chapter 4 the investigation of the toxin profile and content of six strains of Ostreopsis sp. from Cyprus Island is reported. The samples were analyzed through an integrated approach based on molecular, chemical and eco-toxicological methods. Cypriot Ostreopsis sp. was found to be a species distinct from O. cf. ovata and O. cf. siamensis, belonging to the Atlantic/Mediterranean Ostreopsis spp. clade. Some variability in toxin profiles emerged: three strains produced OVTX-a, OVTX-d, OVTX-e and isobaric PLTX, so far found only in O. cf. ovata, the other three strains produced only new PLTX-like compounds, that we named OVTX-i, -j1, -j2, and -k. The new OVTXs had the same carbon skeleton as OVTX-a differing primarily for an additional C2H2O2 moiety and an unsaturation in the region C49-C52. Other minor structural differences were found, including the presence of a hydroxyl group at C44 (in OVTX-j1 and -k) and the lack of a hydroxyl group in the region C53-C78 (in OVTX-i and -j1). Toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06-2.8 pg/cell, definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, eco-toxicological test on A. salina nauplii demonstrated that Ostreopsis sp. presents a very low toxicity compared to O. cf. ovata. The whole of these data suggest that Ostreopsis sp. from Cyprus Island pose a relatively low risk to humans. Chapter 5 reports the chemical analysis of five strains of Ostreopsis spp. collected from Lebanon coasts and of six strains of Ostreopsis spp. (ovata, siamensis and Ostreopsis sp.) from New Zealand and Australia. LC-HRMS of Lebanese extracts showed that three strains produced OVTX-a, OVTX-d, OVTX-e in very minute amounts (0.28-0.94 pg/cell), thus presentig a low risk to humans. The toxin profile of these strains quali-quantitatively matched with that of other Ostreopsis sp. strains from Cyprus Island. The other extracts did not contain any PLTX congener. Molecular analyses showed that Ostreopsis sp. from Lebanon and from Cyprus Island are actually the same species that we named Ostreopsis fattorussoi. LC-HRMS analysis of Ostreopsis spp. strains from New Zealand and Australia showed that none of the known PLTX congeners so far known were contained in any of the extracts. Only a new analogue, was present in one of the Ostreopsis sp. extracts. It presented a C5H7ON moiety as well as 2 unsaturation less than OVTX-a. An extensive LC-HRMSn study, allowed to locate such modification in the very limited region stretching from the A-side terminal to C-8. This compound presented a characteristic fragmentation behavior: indeed, a formaldehyde loss could be observed from the precursor ion as well as from most of the A-side fragment ions. Formaldehyde losses may occur when a CH2OH moiety is present in the molecule following a Mac Lafferty-like rearrangement. We located the CH2OH moiety on the terminal amide. Chapter 6 reports on a survey of phytoplankton and algal toxins in Nigerian coastal waters. Seawater samples were obtained from four sites for phytoplankton identification (Bar Beach and Lekki in Lagos State, Port Harcourt in Rivers State and Uyo in Akwa Ibom State), on three occasions between the middle of October 2014 and the end of February 2015. The phytoplankton community was generally dominated by diatoms and cyanobacteria; however, several species of dinoflagellates were also identified: Dinophysis caudata, Lingulodinium polyedrum and two benthic species of Prorocentrum. Passive samplers (containing Diaion® HP-20 resin) were deployed for several 1-week periods on the same four sites to obtain profiles of algal toxins present in the seawater. Quantifiable amounts of OA and PTX-2, as well as traces of DTX-1 were detected at several sites. The highest toxin concentrations (60 ng OA g/HP-20 resin) were found at Lekki and Bar Beach stations, which had also the highest salinities. Non-targeted analysis using full-scan HRMS showed that algal metabolites differed from site to site and for different sampling occasions. Screening against a marine natural products database indicated the potential presence of cyanobacterial compounds in the water column, which was also consistent with phytoplankton analysis. Chapter 7 reports another application of passive samplers in the French lagoon of Ingril. Seawater portions of 30 L were collected and pre concentrated by passive sampling with HP-20 resin over a 48 h period. Detection of lipophilic toxins in the extracts of the resin was carried out using liquid chromatography coupled to tandem mass spectrometry. This combination allowed the detection of sub-ppb levels of dissolved toxins and would permit further studies for accurate modelling of the toxins adsorption behavior by passive sampling devices. In particular, we determined the levels of OA, DTX-1, PTX-2 and PnTX-G in seawater from Ingril lagoon. OA was the most concentrated compound with ca. 8.6 ng/L, followed by DTX-1 with ca. 1.4 ng/L, and both PTX-2 and PnTX-G at ca. 0.2-0.3 ng/L. This is one of the first direct analyses of lipophilic dinoflagellate toxins in seawater. However, these concentrations were observed in a lagoon and should be confirmed in open coastal waters. Chapter 8 describes preliminary studies aiming to quantitative isolation of palytoxin. Previous reported isolation procedure, provided very poor recoveries of palytoxin congeners as pure compound, with no indication whether such low recoveries were due to instability of the compounds in solution or to irreversible adsorption to materials or to other uncontrolled factors. In view of a large scale isolation work aimed to preparation of PLTX reference material, the causes underlying the huge PLTX loss were investigated. We focused on the evaporation of PLTX under various experimental conditions namely the use of different evaporation systems (Centrifugal Vacuum Concentrator and N2 stream) versus freeze drying, complete drying versus concentration, the influence of various solvents (aqueous or pure organic), the effect of most common materials (normal and silanized glass vials, polypropylene and Teflon tubes) and of the re-dissolution solvent (nature and quantity) on toxin recovery. Preliminary results on stability of PLTX under various acidic conditions were also obtained. PLTX behaved differently when it was simply concentrated or completely dried down. Recoveries were strongly dependent on solubility of PLTX in the mixtures used as well as on the materials in which evaporation was carried out. We found that, in order to enhance PLTX recovery, freeze drying is an appropriate procedure to be avoided. In general, the highest recoveries were obtained when PLTX was completely dried down in Teflon carrying out evaporation in aqueous blends or concentration in pure organic solvent. The worst recoveries were obtained using glass materials probably due to the sticking of PLTX on the surface of the vials; in order to reduce this phenomenon water should be used in the mixture. Experiments on stability of PLTX in acid blends demonstrated that PLTX is an acid-sensitive molecule and that, depending on pH, it rapidly degradates forming mainly a PLTX methyl-ester by cleavage of the enamide functionality contained in the A-side terminal of the molecule. Structural insights on the PLTX methyl-ester were gained by LC-HRMS/MS