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A quantitative study on the sponge spicules present in a series of horizontal core samples, taken at different levels, from a 1.5 m thick living matte within the Posidonia oceanica meadow of Prelo Bay (Eastern Ligurian Sea), has been performed. Sponge spicule amounts were evaluated by microscopical and chemical analyses. From the superficial samples the spicule number decreased until 60–80 cm depth to increase progressively until the deepest studied layer, 140 cm depth. The same results were obtained from the trend of spicule volume and biogenic silica tested by chemical analysis. Among the recognisable spicule types, the most common are oxeas, followed by tylostyles, achantostyles and strongyles. However, oxeas, tylostyles and achantostyles showed the same trend of the entire spicule population, whereas strongyles had an opposite trend with a maximum value at the 80–100 cm depth. According to the hypothesis that the amount of spicules in the sediment is proportional or at least related to the sponge biomass existing in the surrounding area, our data suggest that the sponge assemblage of Prelo Bay should not have been constant in the past 100–200 years. Our hypothesis is that the rapid urban and industrial development that occurred along the Ligurian coast after the Second World War, particularly in the 1960s, had an important impact on the sponge assemblages of the area. [ABSTRACT FROM AUTHOR]

Several Dinophysis species can produce potent lipophilic toxins that pose a risk to human health when contaminated seafood is consumed, especially filter-feeding bivalve mussels. In the mussel farms of the Northwestern Adriatic Sea, seawater and seafood are regularly monitored for the presence of Dinophysis species and their associated toxins, but the current methodological approaches, such as light microscopy determinations, require a long time to make results available to local authorities. A molecular qPCR-based assay can be used to quantify various toxic Dinophysis species in a shorter timeframe. However, this approach is not currently employed in official testing activities. In this study, field samples were collected monthly or bi-weekly over one year from various mussel farms along the Northwestern Adriatic coast. The abundance of Dinophysis species in the seawater was determined using both traditional microscopy and qPCR assays. In addition, the concentration of lipophilic toxins for DSP in mussel flesh was quantified using LC-MS/MS focusing on the okadaic acid group. Dinophysis spp. site-specific single cells were isolated and analysed by qPCR yielding a mean rDNA copy number per cell of 1.21 × 10 4 ± 1.81 × 10 3 . The qPCR assay gave an efficiency of 98 % and detected up to 10 copies of the rDNA target gene. The qPCR and light microscopy determinations in environmental samples showed a significant positive correlation (Spearman r s = 0.57, p-value < 0.001) with a ratio of 2.24 between the two quantification methods, indicating that light microscopy estimates were generally 44.6 % lower than those obtained by the qPCR assay. The qPCR approach showed several advantages such as rapidity, sensitivity and efficiency over conventional microscopy analysis, showing its potential future role in phytoplankton monitoring under the Official Controls Regulations for shellfish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The dynamics of hydrographic and biogeochemical properties in a Northwestern coastal area of the Adriatic Sea were investigated. The time series data from continuous observation (2007-2022) allowed the investigation of annual trends and seasonal cycles along a coastal transect influenced by local river discharge. Various statistical models were used to investigate water temperature, salinity, chlorophyll a, dissolved organic, inorganic and particulate nutrients, precipitation and river discharge. It was found that the local river discharge regime played an essential role in interannual, and seasonal biogeochemical dynamics associated with global climate change in the Mediterranean region. A significant trend towards oligotrophic conditions was detected, as evidenced by the downward trend in the river mouth and on the sea of chlorophyll a (-0.2 μg L -1 in the sea), dissolved organic and inorganic nitrogen and phosphorus (i.e., -0.43 μM yr -1 of DON in the sea and -6.67 of DIN μM yr -1 in the river mouth or -0.07 μM yr -1 of DOP and -0.02 μM yr -1 of DIP in the river mouth) and silicate (-2.47 μM yr -1 in the river mouth) concentrations. Salinity showed a long-term increase in the sea (0.08 yr -1 ), corresponding to a significant decrease in water discharge from the local river (-0.27 m 3  s -1 yr -1 ) and precipitation (-0.06 mm yr -1 ). The dissolved organic and inorganic nutrients highlighted a different seasonal accumulation under the river runoff regime. The nutrient enrichment was predominantly driven by river contribution. Data analysis showed that the coastal biogeochemical properties dynamics were mostly influenced by river discharge and precipitation regimes, which in turn are driven by climate change variability in the North-western Adriatic Sea., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

This study is based on assessing fecal indicator bacteria contamination along meteorological, hydrological and physical-chemical variables after high rainy events during the summer period. The study focused on four different coastal sites in the western and eastern Adriatic coast characterized by various geomorphological and hydrological features, levels of urbanization and anthropogenic pressures, with the aim of finding appropriate and effective solutions to ensure the safety and sustainability of tourism and public health. Detailed in-situ survey revealed a wide range of fecal indicator bacterial (FIB) across the different river mouths with concentrations of E. coli ranging from 165 to 6700 CFU 100 mL -1 . It was found that nitrogen compounds track microbial load and acted as tracers for fecal contaminants. Further, a modelling tool was also used to analyze the spatial and temporal distribution of fecal pollution at these coastal sites. The integrated monitoring through high frequent survey in river waters and modeling framework allowed for the estimation of fecal indicator bacterial load at the river mouth and examination of fecal pollutant dispersion in recreational waters, considering different scenarios of fecal dispersion along the coast. This study formed the basis of a robust decision support system aimed at improving the management of recreational areas and ensuring the protection of water bodies through efficient management of bathing areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Marine toxins have a significant impact on seafood resources and human health. Up to date, mainly based on bioassays results, two genera of toxic microalgae, Gambierdiscus and Fukuyoa have been hypothesized to produce a suite of biologically active compounds, including maitotoxins (MTXs) and ciguatoxins (CTXs) with the latter causing ciguatera poisoning (CP) in humans. The global ubiquity of these microalgae and their ability to produce (un-)known bioactive compounds, necessitates strategies for screening, identifying, and reducing the number of target algal species and compounds selected for structural elucidation. To accomplish this task, a dereplication process is necessary to screen and profile algal extracts, identify target compounds, and support the discovery of novel bioactive chemotypes. Herein, a dereplication strategy was applied to a crude extract of a G. balechii culture to investigate for bioactive compounds with relevance to CP using liquid chromatography-high resolution mass spectrometry, in vitro cell-based bioassay, and a combination thereof via a bioassay-guided micro-fractionation. Three biologically active fractions exhibiting CTX-like and MTX-like toxicity were identified. A naturally incurred fish extract (Sphyraena barracuda) was used for confirmation where standards were unavailable. Using this approach, a putative I/C-CTX congener in G. balechii was identified for the first time, 44-methylgambierone was confirmed at 8.6 pg cell -1 , and MTX-like compounds were purported. This investigative approach can be applied towards other harmful algal species of interest. The identification of a microalgal species herein, G. balechii (VGO920) which was found capable of producing a putative I/C-CTX in culture is an impactful advancement for global CP research. The large-scale culturing of G. balechii could be used as a source of I/C-CTX reference material not yet commercially available, thus, fulfilling an analytical gap that currently hampers the routine determination of CTXs in various environmental and human health-relevant matrices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plastic pollution is a global issue posing a threat to marine biota with ecological implications on ecosystem functioning. Micro and nanoplastic impact on phytoplankton autotrophic species (e.g., cell growth inhibition, decrease in chlorophyll a and photosynthetic efficiency and hetero-aggregates formation) have been largely documented. However, the heterogeneity of data makes rather difficult a comparison based on size (i.e. micro vs nano). In addition, knowledge gaps on the ecological impact on phytoplankton assemblage structure and functioning are evident. A new virtual meta-analysis on cause-effect relationships of micro and nanoplastics on phytoplankton species revealed the significant effect posed by polymer type on reducing cell density for tested PVC, PS and PE plastics. Linked with autotrophic phytoplankton role in atmospheric CO 2 fixation, a potential impact of plastics on marine carbon pump is discussed. The understanding of the effects of microplastics and nanoplastics on the phytoplankton functioning is fundamental to raise awareness on the overall impact on the first level of marine food web. Interactions between micro and nanoplastics and phytoplankton assemblages have been quite documented by in vitro examinations; but, further studies considering natural plankton assemblages and/or large mesocosm experiments should be performed to evaluate and try predicting ecological impacts on primary producers., (Copyright © 2021. Published by Elsevier Ltd.)

In the Adriatic Sea, massive rainfall events are causing flooding of rivers and streams, with severe consequences on the environment. The consequent bacterial contamination of bathing water poses public health risks besides damaging tourism and the economy. This study was conducted in the framework of WATERCARE, an EU Interreg Italy-Croatia Project, which aims at reducing the impact of microbial contamination on Adriatic bathing water due to heavy rainfall events drained in the local sewage network and; enhancing the quality of local waters; and providing support for the decision-making processes regarding the management of bathing water in line with EU regulations. The study involved the development of an innovative water quality integrated system that helps meet these objectives. It consists of four components: a real time hydro-meteorological monitoring system; an autosampler to collect freshwater samples during and after significant rainfall events; a forecast system to simulate the dispersion of pollutants in seawater; and a real-time alert system that can predict the potential ecological risk from the microbial contamination of seawater. The system was developed and tested at a pilot site (Fano, Italy). These preliminary results will be used to develop guidelines for urban wastewater and coastal system quality assessments to contribute to develop policy actions and final governance decisions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Marine diatoms have been identified among the most abundant taxa of microorganisms associated with plastic waste collected at sea. However, the impact of nano-sized plastic fragments (nanoplastics) at single cell and population level is almost unknown. We exposed the marine diatom Skeletonema marinoi to model polystyrene nanoparticles with carboxylic acid groups (PS-COOH NPs, 90 nm) for 15 days (1, 10, 50 μg/mL). Growth, reactive oxygen species (ROS) production, and nano-bio-interactions were investigated. No effect on diatom growth was observed, however Dynamic light scattering (DLS) demonstrated the formation of large PS aggregates which were localized at the diatoms' fultoportula process (FPP), as shown by TEM images. Increase production of ROS and reduction in chain length were also observed upon PS NPs exposure (p < 0.005). The observed PS-diatom interaction could have serious consequences on diatoms ecological role on the biogeochemical cycle of carbon, by impairing the formation of fast-sinking aggregates responsible for atmospheric carbon fixation and sequestration in the ocean sea floor. S. marinoi exposure to PS NPs caused an increase of intracellular and extracellular oxidative stress, the reduction of diatom's chain length and the adhesion of PS NPs onto the algal surface., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Plastics are the most abundant marine debris globally dispersed in the oceans and its production is rising with documented negative impacts in marine ecosystems. However, the chemical-physical and biological interactions occurring between plastic and planktonic communities of different types of microorganisms are poorly understood. In these respects, it is of paramount importance to understand, on a molecular level on the surface, what happens to plastic fragments when dispersed in the ocean and directly interacting with phytoplankton assemblages. This study presents a computer-aided analysis of electron paramagnetic resonance (EPR) spectra of selected spin probes able to enter the phyoplanktonic cell interface and interact with the plastic surface. Two different marine phytoplankton species were analyzed, such as the diatom Skeletonema marinoi and dinoflagellate Lingulodinium polyedrum, in absence and presence of polyethylene terephthalate (PET) fragments in synthetic seawater (ASPM), in order to in-situ characterize the interactions occurring between the microalgal cells and plastic surfaces. The analysis was performed at increasing incubation times. The cellular growth and adhesion rates of microalgae in batch culture medium and on the plastic fragments were also evaluated. The data agreed with the EPR results, which showed a significant difference in terms of surface properties between the diatom and dinoflagellate species. Low-polar interactions of lipid aggregates with the plastic surface sites were mainly responsible for the cell-plastic adhesion by S. marinoi, which is exponentially growing on the plastic surface over the incubation time., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Increased anthropic pressure on the coastal zones of the Mediterranean Sea caused an enrichment in nutrients, promoting microalgal proliferation. Among those organisms, some species, such as the dinoflagellate Alexandrium minutum, can produce neurotoxins. Toxic blooms can cause serious impacts to human health, marine environment and economic maritime activities at coastal sites. A mathematical model predicting the presence of A. minutum in coastal waters of the NW Adriatic Sea was developed using a Random Forest (RF), which is a Machine Learning technique, trained with molecular data of A. minutum occurrence obtained by molecular PCR assay. The model is able to correctly predict more than 80% of the instances in the test data set. Our results showed that predictive models may play a useful role in the study of Harmful Algal Blooms (HAB).

Paralytic shellfish toxins (PST) and tetrodotoxin (TTX) are naturally-occurring toxins that may contaminate the food chain, inducing similar neurological symptoms in humans. They are co-extracted under the same conditions and thus their combined detection is desirable. Whilst PST are regulated and officially monitored in Europe, more data on TTX occurrence in bivalves and gastropods are needed before meaningful regulations can be established. In this study, we used three separate analytical methods - pre-column oxidation with liquid chromatography and fluorescence detection, ultrahigh performance hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (MS/MS) and HILIC high resolution (HR) MS/MS - to investigate the presence of PST and TTX in seawater and shellfish (mussels, clams) collected in spring summer 2015 to 2017 in the Mediterranean Sea. Samples were collected at 10 sites in the Syracuse Bay (Sicily, Italy) in concomitance with a mixed bloom of Alexandrium minutum and A. pacificum. A very high PST contamination in mussels emerged, unprecedentedly found in Italy, with maximum total concentration of 10851 μg saxitoxin equivalents per kg of shellfish tissue measured in 2016. In addition, for the first time TTX was detected in Italy in most of the analysed samples in the range 0.8-6.4 μg TTX eq/kg. The recurring blooms of PST-producing species over the 3-year period, the high PST levels and the first finding of TTX in mussels from the Syracuse bay, suggest that monitoring programmes of PST and TTX in seafood should be activated in this geographical area., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Plastic debris carry fouling a variety of class-size organisms, among them harmful microorganisms that potentially play a role in the dispersal of allochthonous species and toxic compounds with ecological impacts on the marine environment and human health. We analyzed samples of marine plastics floating at the sea surface using a molecular qPCR assay to quantify the attached microalgal taxa, in particular, harmful species. Diatoms were the most abundant group of plastic colonizers with maximum abundance of 8.2 × 10 4  cells cm -2 of plastics, the maximum abundance of dinoflagellates amounted to 1.1 × 10 3  cells cm -2 of plastics. The most abundant harmful microalgal taxon was the diatom Pseudo-nitzschia spp., including at least 12 toxic species, and the dinoflagellate Ostreopsis cf. ovata with 6606 and 259 cells cm -2 , respectively. The abundance of other harmful microalgal species including the toxic allochthonous dinoflagellate Alexandrium pacificum ranged from 1 to 73 cells cm -2 . In the present study, a direct relationship between the abundance of harmful algal species colonizing the plastic substrates and their toxin production was found. The levels of potential toxins on plastic samples ranged from 10 1 to 10 2  ng cm -2 , considering the various toxin families produced by the colonized harmful microalgal species. We also measured the rate of adhesion by several target microalgal species. It ranged from 1.8 to 0.3 day -1 demonstrating the capacity of plastic substrate colonizing rapidly by microalgae. The present study reports the first estimates of molecular quantification of microorganisms including toxin producing species that can colonize plastics. Such findings provide important insights for improving the monitoring practice of plastics and illustrate how the epi-plastic community can exacerbate the harmful effects of plastics by dispersal, acting as an alien and toxic species carrier and potentially being ingested through the marine trophic web., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The silicon transport and use inside cells are key processes for understanding how diatoms metabolize this element in the silica biogenic cycle in the ocean. A spin-probe electron paramagnetic resonance (EPR) study over time helped to investigate the interacting properties and the internalization mechanisms of silicic acid from different silicon sources into the cells. Diatom cells were grown in media containing biogenic amorphous substrates, such as diatomaceous earth and sponge spicules, and crystalline sodium metasilicate. It was found that the amorphous biogenic silicon slowed down the internalization process probably due to formation of colloidal particles at the cell surface after silicic acid condensation. Weaker interactions occurred with sponge spicules silicon source if compared to the other sources. The EPR results were explained by analyzing transcript level changes of silicon transporters (SITs) and silaffins (SILs) in synchronized Thalassiosira pseudonana cultures over time. The results indicated that the transport role of SITs is minor for silicic acid from both biogenic and crystalline substrates, and the role of SIT3 is linked to the transport of silicon inside the cells, mainly in the presence of sponge spicules. SIL3 transcripts were expressed in the presence of all silicon sources, while SIL1 transcripts only with sponge spicules. The data suggest that the transport of silicic acid from various silicon sources in diatoms is based on different physico-chemical interactions with the cell surface., (Copyright © 2017 Elsevier B.V. All rights reserved.)

During the past decade, next generation sequencing (NGS) technologies have provided new insights into the diversity, dynamics, and metabolic pathways of natural microbial communities. But, these new techniques face challenges related to the genome size and level of genome complexity of the species under investigation. Moreover, the coverage depth and the short-read length achieved by NGS based approaches also represent a major challenge for assembly. These factors could limit the use of these high-throughput sequencing methods for species lacking a reference genome and characterized by a high level of complexity. In the present work, the evolutionary history, mainly consisting of gene transfer events from bacteria and unicellular eukaryotes to microalgae, including harmful species, is discussed and reviewed as it relates to NGS application in microbial communities, with a particular focus on harmful algal bloom species and dinoflagellates. In the context of genetic population studies, genotyping-by-sequencing (GBS), an NGS based approach, could be used for the discovery and analysis of single nucleotide polymorphisms (SNPs). The NGS technologies are still relatively new and require further improvement. Specifically, there is a need to develop and standardize tools and approaches to handle large data sets, which have to be used for the majority of HAB species characterized by evolutionary highly dynamic genomes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The new benthic toxic dinoflagellate, Ostreopsis fattorussoi sp. nov., is described from the Eastern Mediterranean Sea, Lebanon and Cyprus coasts, and is supported by morphological and molecular data. The plate formula, Po, 3', 7″, 6c, 7s, 5‴, 2'''', is typical for the Ostreopsis genus. It differs from all other Ostreopsis species in that (i) the curved suture between plates 1' and 3' makes them approximately hexagonal, (ii) the 1' plate lies in the left half of the epitheca and is obliquely orientated leading to a characteristic shape of plate 6″. The round thecal pores are bigger than the other two Mediterranean species (O. cf. ovata and O. cf. siamensis). O. fattorussoi is among the smallest species of the genus (DV: 60.07 ± 5.63 μm, AP: 25.66 ± 2.97 μm, W: 39.81 ± 5.05 μm) along with O. ovata. Phylogenetic analyses based on the LSU and internal transcribed spacer rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. Ostreopsis fattorussoi produces OVTX-a and structural isomers OVTX-d and -e, O. cf. ovata is the only other species of this genus known to produce these toxins. The Lebanese O. fattorussoi did not produce the new palytoxin-like compounds (ovatoxin-i, ovatoxin-j 1 , ovatoxin-j 2 , and ovatoxin-k) that were previously found in O. fattorussoi from Cyprus. The toxin content was in the range of 0.28-0.94 pg · cell -1 . On the Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18°C-31.5°C), with peaks in June and August., (© 2016 Phycological Society of America.)

Blooms of benthic dinoflagellates of the genus Ostreopsis (mainly O. cf. ovata and occasionally O. cf. siamensis) represent a serious concern for humans in the Mediterranean area, due to production of palytoxin-like compounds listed among the most potent marine toxins known. In this work, six strains of Ostreopsis sp. from Cyprus Island 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 ovatoxin-a (OVTX-a), OVTX-d, OVTX-e, and isobaric palytoxin, so far found only in O. cf. ovata; the other three strains produced only new palytoxin-like compounds, which we named ovatoxin-i, ovatoxin-j1, ovatoxin-j2, and ovatoxin-k. The new ovatoxins present the same carbon skeleton as ovatoxin-a, differing primarily in 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 OVTX-k) and the lack of a hydroxyl group in the region C53-C78 (in OVTX-i and OVTX-j1). The toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06-2.8 pg cell(-1), definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, an eco-toxicological test on Artemia 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 poses a relatively low risk to humans.

Paralytic shellfish poisoning (PSP) is a serious human illness caused by the ingestion of seafood contaminated with saxitoxin and its derivatives (STXs). These toxins are produced by some species of marine dinoflagellates within the genus Alexandrium. In the Mediterranean Sea, toxic Alexandrium spp. blooms, especially of A. minutum, are frequent and intense with negative impact to coastal ecosystem, aquaculture practices and other economic activities. We conducted a large scale study on the sxt gene and toxin distribution and content in toxic dinoflagellate A. minutum of the Mediterranean Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques. We developed a new qPCR assay for the estimation of the sxtA1 gene copy number in seawater samples during a bloom event in Syracuse Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 × 10° sxtA1 gene copy number per reaction. The linear correlation between sxtA1 gene copy number and microalgal abundance and between the sxtA1 gene and STX content allowed us to rapidly determine the STX-producing cell concentrations of two Alexandrium species in environmental samples. In these samples, the amount of sxtA1 gene was in the range of 1.38 × 10(5) - 2.55 × 10(8) copies/L and the STX concentrations ranged from 41-201 nmol/L. This study described a potential PSP scenario in the Mediterranean Sea.

A molecular PCR-based assay was developed and applied to macrophyte and seawater samples containing mixed microphytobenthic and phytoplanktonic assemblages, respectively, in order to detect toxic Ostreopsis species in Mediterranean Sea. The specificity and sensitivity of the molecular PCR assay were assessed with both plasmidic and genomic DNA of the target genus or species using taxon-specific primers in the presence of background macrophyte DNA. The PCR molecular technique allowed rapid detection of the Ostreopsis cells, even at abundances undetectable within the resolution limit of the microscopy technique. Species-specific identification of Ostreopsis was determined only by PCR-based assay, due to the inherent difficulty of morphological identification in field samples. In the monitoring of the toxic Ostreopsis blooms PCR-based methods proved to be effective tools complementary to microscopy for rapid and specific detection of Ostreopsis and other toxic dinoflagellates in marine coastal environments., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The mucilage phenomenon, a sporadic but massive accumulation of gelatinous material, can cause serious damage to the tourism and fishing industries along the Adriatic coast. Mucilage is presently thought to be the result of the aggregation of dissolved organic matter (DOM) into particulate organic matter (POM). Three principal classes of compounds have been identified in organic matter by spectrometric determination: carbohydrates, proteins and lipids. Carbohydrates are suspected to play a role in the first steps of DOM aggregation. Despite its importance in understanding the processes leading to mucilage formation, our present knowledge of the composition of the mucilage carbohydrate fraction is incomplete. Due to its high sensitivity and specificity, liquid chromatography coupled with electrospray-ionization tandem mass spectrometry (LC-ESIMS/MS) is gaining an increasing importance as a powerful technique for carbohydrate purification and characterization in complex samples. In this work, LC-ESIMS/MS is proposed as a useful method for the investigation of the oligosaccharide content in mucilage samples. The approach was applied using 3-7 unit maltooligosaccharides as reference compounds. The composition of the investigated mucilage sample was further investigated combining LC-ESIMS/MS with classic approaches, such as spectroscopic techniques and liquid chromatography coupled with the refractory index LC-RI.

Carbohydrate contents in seawater, mucilage, and mucilage interstitial waters were analyzed during episodes of mucilage formation in the summers of 2000 and 2001 in the northern Adriatic Sea off Pesaro and in the Gulf of Trieste using 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) and 2,4,6-tripyridyl-s-triazine (TPTZ) assays. The significant presence of polysaccharides in seawater in the presence of mucilage has an important impact on the agglomeration processes forming gelatinous material (macrogels). Characterization of oligosaccharides in the water-soluble fraction of mucilage using HPLC/RI revealed maltose and pentaose as the main components.

Numerous studies have demonstrated a decrease in glucose-6-phosphate dehydrogenase (G6PD) activity during aging in many cell types, including red blood cells, fibroblasts and lens cells. Moreover, the intracellular activity of G6PD has been shown to be regulated by binding to cell organelles. To investigate whether binding of G6PD to cell organelles is related with the decrease in its activity during aging, distribution patterns of G6PD activity and protein were assessed in small (SI) and large (LI) intestine of 3-month-old and 28-month-old rats. Enzyme activity, as measured spectrophotometrically, did not show any significant change with aging in SI or LI. Enzyme histochemistry, performed by subtracting activity staining of 6-phosphogluconate dehydrogenase (6PGD) from that of G6PD, showed a lower net G6PD activity in SI and LI epithelium of old rats in comparison with young rats. G6PD activity did not change significantly with aging in the muscularis externa of SI and LI. Immunoelectron microscopic analysis of G6PD protein allowed us to measure the density of G6PD molecules in cellular compartments, and the fraction of enzyme bound to cell organelles. In SI and LI epithelia, density of G6PD molecules was higher in old rats than in young rats; however, the fraction of enzyme bound to cell organelles also increased with aging. These data suggest that G6PD activity in epithelium of SI and LI decreases with aging due to the accumulation of significant amounts of enzyme bound to cell organelles, a condition which makes it less active than the soluble enzyme.

Glucose-6-phosphate dehydrogenase (G6PD) purified from rabbit brain is composed of two identical subunits of 56 kDa. The enzyme exhibits biphasic pH curve, linear Arrhenius plot and elevated susceptibility to inactivation by metal catalyzed oxidation and thiol binding reagents. 4-Hydroxy-2-nonenal (HNE) is able to inactivate the enzyme after only a few minutes of incubation. Since reactive oxygen species and G6PD-HNE adducts form easily in brain under conditions of oxidative stress, these findings have important implications in the loss of active G6PD molecules in vivo, a process which lowers the antioxidant protection and may be critical for neuron survival.

Lingual epithelium undergoes oxidative stress and apoptosis with consequent renewal of superficial keratinized cells by proliferation and differentation of the stem cells of the basal germinative layer. In 3 distinct areas of lingual epithelium of rat and rabbit, the anterior third, central third and posterior third, we determined the activity of hexose monophosphate shunt enzymes and antioxidant enzymes, which are essential for support of cell proliferation and differentation. Enzymatic assays of the epithelium showed that glucose-6-phosphate dehydrogenase (G6PD) activity was highest in the anterior third, whereas activity of glutathione peroxidase, 6-phosphogluconate dehydrogenase, glutathione reductase, superoxide dismutase and catalase was similar over all areas. Histochemical localization of activity and immunohistochemical localization of protein of G6PD showed that all types of papillae had a similar G6PD content; moreover, the presence of different G6PD isoforms in the 3 areas was excluded by electrophoretic analysis. We conclude that the higher G6PD activity in the anterior part of the epithelium is due only to the anatomical organization of the epithelial surface of this area, in which many filiform and fungiform papillae are arranged in a compact manner, which corresponds with a higher number of proliferating and differentiating cells. These processes need products of G6PD activity. This study indicates that G6PD is a good marker for the number of differentiating cells in tongue epithelium.