A Hotspot of TTX Contamination in the Adriatic Sea: Study on the Origin and Causative Factors.

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin's origin and the way through which it accumulates in mussels. A two-year study (spring-summer 2020-2021) confirmed the recurrent presence of TTX (11-68 µg kg-1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9-296 µg kg-1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a "potential vector" should therefore be further investigated.

Morphological Variability of Pseudo-nitzschia pungens Clade I (Bacillariophyceae) in the Northwestern Adriatic Sea.

Pseudo-nitzschia pungens is a common component of the phytoplankton community of the northern Adriatic Sea. In this study, an in-depth morphological analysis of P. pungens was carried out in both cultured strains isolated in different periods and field samples, revealing a surprisingly wide variability in a number of details, with both the gross morphology and ultrastructural levels deviating from the nominal P. pungens. Colonies showed an overlap (from one-third to one-sixth) and a transapical axis (rarely reaching 3 µm), strongly differing from the original description of the species. Moreover, valves may be either symmetrical or slightly asymmetrical, with striae almost always biseriate but sometimes uniseriate or triseriate. Poroids' morphology in cingular bands was characterized by a wide variability (square, circular, or rectangular poroids without or with up to two hymen sectors), with several combination of them, even within the same cingular band. Phylogenetic analyses based on ITS rDNA showed that the P. pungens of the northern Adriatic Sea belonged to clade I. Domoic acid was not detected.

Distribution and potential toxicity of benthic harmful dinoflagellates in waters of Florida Bay and the Florida Keys.

Few studies have been carried out on benthic dinoflagellates along the Florida Keys, and little is known about their distribution or toxicity in Florida Bay. Here, the distribution and abundance of benthic dinoflagellates was explored in northern and eastern Florida Bay and along the bay and ocean sides of the Florida Keys. Isolates were brought into culture and their toxicity was tested with oyster larvae bioassays. Seven genera were detected, including Prorocentrum, Coolia, Ostreopsis, Amphidinium, Gambierdiscus, Fukuyoa (all included potentially toxic species) and Sinophysis. In general, distribution increased with water temperature and nutrient availability, especially that of phosphate. This study documented the first record of Coolia santacroce in the Florida Keys. Potential toxic effects of Gambierdiscus caribaeus, the abundance of which exceeded 1000 cells g-1 fw at some sites, were established using oyster larvae as a bioassay organism. These findings suggest a potential risk of ciguatera fish poisoning in this area.

Role of temperature and nutrients on the growth and toxin production of Prorocentrum hoffmannianum (Dinophyceae) from the Florida Keys.

The benthic dinoflagellate Prorocentrum hoffmannianum M.A. Faust is typical of tropical warm waters and produces biotoxins responsible for diarrhetic shellfish poisoning (DSP). In this study, the effect of temperature and nutrient limitation on growth and toxin production of P. hoffmannianum isolated from field samples collected in the Florida Keys was investigated. Batch culture experiments were ran at two temperatures (i.e. 21 ± 0.1 and 27 ± 0.1 °C) and under nitrogen-limited (14.7 µmol L-1 N-NO3- and 18.1 µmol L-1 P-PO43-) and phosphorus-limited (441 µmol L-1 N-NO3- and 0.6 µmol L-1 P-PO43-) levels with respect to control nutrient conditions (441 µmol L-1 N-NO3-and 18.1 µmol L-1 P-PO43-). Both temperature and nutrient conditions significantly affected growth rates and maximum yield of P. hoffmannianum with the maximum values being recorded at the higher temperature and in the replete medium. Production of okadaic acid was induced under all conditions (from 13.5 to 859.8 pg cell-1), with values up to one order of magnitude higher than those observed in other DSP toxin producing species. Toxin production was enhanced under P limitation at 27 °C, corroborating the theory that toxin production is modulated by cell physiological conditions, which are in turn affected by a wide spectrum of factors, including environmental stressors such as nutrient availability. Toxin fraction released in the growth medium was negligible. No okadaic acid esters were detected in this strain of P. hoffmannianum.

Plankton dynamics across the freshwater, transitional and marine research sites of the LTER-Italy Network. Patterns, fluctuations, drivers.

A first synoptic and trans-domain overview of plankton dynamics was conducted across the aquatic sites belonging to the Italian Long-Term Ecological Research Network (LTER-Italy). Based on published studies, checked and complemented with unpublished information, we investigated phytoplankton and zooplankton annual dynamics and long-term changes across domains: from the large subalpine lakes to mountain lakes and artificial lakes, from lagoons to marine coastal ecosystems. This study permitted identifying common and unique environmental drivers and ecological functional processes controlling seasonal and long-term temporal course. The most relevant patterns of plankton seasonal succession were revealed, showing that the driving factors were nutrient availability, stratification regime, and freshwater inflow. Phytoplankton and mesozooplankton displayed a wide interannual variability at most sites. Unidirectional or linear long-term trends were rarely detected but all sites were impacted across the years by at least one, but in many case several major stressor(s): nutrient inputs, meteo-climatic variability at the local and regional scale, and direct human activities at specific sites. Different climatic and anthropic forcings frequently co-occurred, whereby the responses of plankton communities were the result of this environmental complexity. Overall, the LTER investigations are providing an unparalleled framework of knowledge to evaluate changes in the aquatic pelagic systems and management options.

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.

Ostreopsis fattorussoi sp. nov. (Dinophyceae), a new benthic toxic Ostreopsis species from the eastern Mediterranean Sea.

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-j1 , ovatoxin-j2 , 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.

Effects of the bloom of harmful benthic dinoflagellate Ostreopsis cf. ovata on the microphytobenthos community in the northern Adriatic Sea.

Composition and temporal variation of the microphytobenthos communities of the Conero Riviera (northern Adriatic Sea) were investigated in the course of an annual cycle, focusing on their relationships with blooms of the benthic dinoflagellate Ostreopsis cf. ovata. Sampling was carried out from March 2009 to March 2010 on undisturbed benthic substrata (macroalgae and pebbles). Samples for the study of microphytobenthos were collected with a monthly frequency, while those for the study of Ostreopsis bloom weekly. Benthic diatoms dominated the microphytobenthos communities for most of the annual cycle (except the summer), both in terms of abundance and biomass. In summer, cyanobacteria were dominant (54.04±9.18 and 24.29±11.11% of total abundance and biomass, respectively), while benthic dinoflagellates were an important component of the community in terms of biomass only at the peak of the Ostreopsis bloom in late summer (up to 91% of the total biomass). Among diatoms, the most abundant forms throughout the year were motile species (77.5±3.71% of the population), while erect diatoms formed the majority of the biomass in winter and spring (48.66±16.66 and 48.05±5.56% of total population, respectively). Diatoms were mainly affected by DIN availability, while the patterns of biomass of O. cf. ovata and cyanobacteria were related to salinity and temperature. The biomass of Ostreopsis was also affected by the availability of phosphorus. The results of this study suggest that the proliferation of Ostreopsis affected the structure of the benthic diatom community: motile diatoms were significantly more abundant during the Ostreopsis bloom peak than during the rest of summer, probably because they benefited from the abundant mucilaginous mat covering the benthic substrata. In the course of the O. cf. ovata bloom the diversity of the microphytobenthos was significantly lower than during the rest of the year, suggesting an influence of both the shading produced by the mucous mat and allelopathic compounds possibly produced by O. cf. ovata.

Marine macrophytes as effective lead biosorbents.

Several species of seagrass and marine macrophytes were investigated for their biosorption performance in the removal of lead from aqueous solution. The effect of pH on the equilibrium of the seagrass Cymodocea nodosa as a biosorbent also was studied. It was found that increasing pH increased lead biosorption, with a maximum uptake of approximately 140 mg/g in the range pH 3.3 to 5. Equilibrium data at different pH levels were successfully fitted to competitive equilibrium models. In addition, the seaweeds belonging to different phyla (i.e., Chlorophyta, Heterokontophyta, and Rhodophyta) were studied for the effect of their structure on equilibrium at a constant pH 5. The brown algae (Heterokontophyta) showed the highest potential for lead sorption, with a maximum uptake of 220 mg/g for C. compressa and 140 mg/g for S. lomentaria. The green algae (Chlorophyta) showed lead uptake in the range 40 to 90 mg/g, and the red algae (Rhodophyta) was least effective, with uptake in the range 10 to 40 mg/g.

MORPHOLOGICAL STUDIES OF SOME MARINE MASTOGLOIA (BACILLARIOPHYCEAE) BELONGING TO SECTION SULCATAE, INCLUDING THE DESCRIPTION OF NEW SPECIES.

Epiphytic diatoms on seagrass and seaweed were collected from tropical (e.g., Siladen Island, Celebes Sea, Indonesia and Phú Bài, China Sea, Vietnam), subtropical (e.g., Sharm el-Sheikh, Red Sea, Egypt), and temperate regions (e.g., Patmos Island, Greece) in 2000, 2005, and 2006. Eight species of Mastogloia, belonging to the section Sulcatae, are described mainly through scanning electron microscopy, including two new species to science, M. oculoides and M. sergiana. These species show a differently shaped median depression on the external valve face between the raphe-sternum and the valve margin. Moreover, they lack a developed conopeum or pseudoconopeum, which covers the median depression in other species of the section Sulcatae. This study gives new insights on the ultrastructure of the Mastogloia's valves and provides an update of their current geographical distribution.

Ostreopsis cf. ovata bloom in the northern Adriatic Sea during summer 2009: ecology, molecular characterization and toxin profile.

Intense blooms of the benthic dinoflagellate Ostreopsis cf. ovata have occurred in the northern Adriatic Sea since 2006. These blooms are associated with noxious effects on human health and with the mortality of benthic organisms because of the production of palytoxin-like compounds. The O. cf. ovata bloom and its relationships with nutrient concentrations at two stations on the Conero Riviera (northern Adriatic Sea) were investigated in the summer of 2009. O. cf. ovata developed from August to November, with the highest abundances in September (1.3×10(6) cells g(-1) fw corresponding to 63.8×10(3) cells cm(-2)). The presence of the single O. cf. ovata genotype was confirmed by a PCR assay. Bloom developed when the seawater temperature was decreasing. Nutrient concentrations did not seem to affect bloom dynamics. Toxin analysis performed by high resolution liquid chromatography-mass spectrometry revealed a high total toxin content (up to 75 pg cell(-1)), including putative palytoxin and all the ovatoxins known so far.

Relevance of the dinoflagellate Gonyaulax fragilis in mucilage formations of the Adriatic Sea.

Oceanographic cruises were carried out monthly from June 1999 to July 2002 to follow the mucilage formation process in the Northern Adriatic Sea. Results show that in correspondence with these events the dinoflagellate Gonyaulax fragilis (Schütt) Kofoid was observed both in the water column and within mucilage aggregates. In the water column, increasing abundances were observed from May until July, with values never exceeding 8500 cells l(-1). Much higher densities were observed within superficial gelatinous aggregates (22800-3400000 cells l(-1)). In mucilage samples, a large number of decomposing cells were present, together with abundant alive cells, enveloped in exudates. G. fragilis isolated from mucilage samples was cultured in three different culture media; it was characterized by a low growth rate but it produced a high amount of polysaccharides. The highest yield both in terms of cell number and carbohydrate production was observed in the medium having the highest nitrogen and phosphorus content and the lowest N/P ratio. The monomeric composition of G. fragilis carbohydrates, compared with that of mucilage samples, showed that in both natural and cultured samples galactose was the most abundant sugar; in addition, an overall good correlation, especially between the monomeric carbohydrate composition of G. fragilis grown in f/2 medium and that of a mucilage sample in which this species was present in high density, was observed.

Inter-annual variations of planktonic food webs in the northern Adriatic Sea.

The temporal dynamics of microphytoplankton, microzooplankton and mesozooplankton were monitored over 37 months in the Adriatic Sea in order to identify alterations in the plankton structures, which can lead to, or enhance the production of macro-aggregates, that affected the entire northern basin in summers 2000 and 2002, and to assess any negative effects of mucilage on plankton temporal patterns. Samples were collected monthly, from June 1999 to July 2002, on three transects at 9 stations across the northern and central Adriatic Sea. Besides the high year-to-year variations in abundances and taxonomical composition, plankton communities only showed a clear seasonal succession during 2001, when since April a grazing food web developed and was able to control large sized phytoplankton increase. In spring-summer 2000 and 2002 consumer abundances remained quite low and the dominant mesozooplankton summer species (Penilia avirostris) did not reach its usual summer maximum. The lack of an efficient top control was more evident on the northernmost transect, where generally grazing food web prevails over the microbial one. A large part of the microphytoplankton blooms, although not particularly intense, was exported to the bottom in the particulate phase, where it was processed by bacteria, enhancing the production of refractory dissolved material.

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

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