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The Prorocentrales are a unique group of dinophytes based on several apomorphic traits, but species delimitation is challenging within the group. Prorocentrum triestinum was described by Josef Schiller in 1918 as an important bloomforming species from Trieste (Mediterranean, Adriatic Sea) with a conspicuous asymmetric outline and a small, asymmetrically located subapical spine. All subsequent records under this name fail to conform to Schiller’s original description. These inconsistencies have their origin in John Dodge’s 1975 revision of Prorocentrum, which placed Prorocentrum redfieldii, a more symmetrical, slender species with a long apical spine, into synonymy under P. triestinum. To clarify this confusion, we collected samples at the type locality of P. triestinum in Trieste and established a strain that is morphologically consistent with the protologue and suitable for use in epitypification. Morphology and rRNA sequence data of this strain were compared with four new strains identified as P. redfieldii from the Mediterranean Sea and the North Atlantic Ocean. Cells of P. triestinum had an asymmetric outline in lateral view and a small, dorso-subapical spine. These features, which are readily resolved by light microscopy, were distinct from those of the nearly symmetrical and slender cells of P. redfieldii, which had a long, apically located spine. The species are nevertheless closely related and share an identical architecture of the periflagellar area with a distinctive, largely reduced accessory pore together with a very small platelet 7. This apomorphy clearly differentiates both species from other species of Prorocentrum. Both species differ in their primary rRNA sequences, and ITS and LSU sequence differences will enable them to be distinguished in future meta-barcoding studies. The present study demonstrates that P. triestinum and P. redfieldii are distinct species and thus contributes to a reliable biodiversity assessment of Prorocentrum.

Nanobodies against cell surface antigens of toxic cyanobacteria Microcystis aeruginosa were recovered by whole-cell biopanning of a naïve phage display library of nanobodies. Six unique sequences were identified and three sub-cloned and purified as fusion immunoreagents together with either green fluorescent protein or AviTag to be used for diagnostics. The yields of nanobody constructs were in the range of 5-10 mg/l and their specificity and sensitivity was initially evaluated by immunofluorescence and by fluorescent enzyme-linked immunosorbent assay (ELISA) using fluorescent nanobodies. The ELISA data confirmed the nanobody specificity but showed that the saturation of the fluorescence signal already in the presence of few hundreds of cells limited the dynamic range of the method. As an alternative, Avi-tagged nanobodies were used in combination with streptavidin-linked horseradish peroxidase for developing a diagnostic colorimetric cell ELISA, the limit-of-detection of which was 3.2 and 4.5 cells/ml for the two tested cyanobacteria strains, whereas the linear range of the assay was expanded from 10 to 10,000 cells. The fluorescent nanobodies were finally exploited for quantifying cyanobacteria by thermal lens spectrometry (TLS) that enabled to reach a limit-of-detection of 1.2 cells/ml and provided a linear range of measurement between 0 and 10,000 cells. No cross-reactivity with unrelated microalgae was detected and both colorimetric ELISA and TLS provided a linear range of detection of few logs. The data indicate that nanobodies are suitable capture reagents and that both TLS and colorimetric ELISA are reliable to monitor variations of cyanobacteria populations.

The colloidal organic matter (COM) was isolated from the exudates of three cultured phytoplonkters, namely the chlophyte nanoflagellate Tetraselmis sp., the diatom Chaetoceros socialis and the dinoflagellate Prorocentrum minimum, from the Gulf of Trieste (northern Adriatic Sea). The isolation of COM was performed by ultrafiltration with molecular weight cut-off membranes of 5 kDa and final desalinisation by dialysis. The composition of the COM was characterised using C elemental analysis and 1H NMR spectroscopy and compared with COM isolated from a marine sample from the same area (Gulf of Trieste). By using 1H NMR spectroscopy, it was possible to semi-quantitatively determine the concentrations of the main biochemical constituents present in the COM samples. The results showed that the phytoplankton COM was predominantly composed of polysaccharides, with minor contributions from proteins and especially lipids. Therefore, the phytoplankton COM mainly contributes to the marine COM pool in the polysaccharide fraction and less in the protein and lipid fractions.

At the present, the identification of planktonic species in coastal water is still a time intensive process performed by highly trained personnel that relies either on qPCR or on light microscopy observation and in vitro culturing. Furthermore, the increasing danger represented by Harmful Algal Blooms (HABs) inside phytoplankton community and the recent implementation of the legislation on ballast water management to prevent the introduction of HABs and NIS (Non Indigenous Species) urge the development of faster and reliable diagnostic methods. Immuno-based approaches could fulfil this need provided that the costs for antibody selection and production will be reduced. In this work it is demonstrated for the first time the feasibility to recover nanobodies (VHHs) selective for native surface epitopes of Alexandrium minutum by direct whole cell bio-panning using a pre-immune phage display library. The recombinant nature of VHHs enabled their rapid engineering into eGFP fluorescent reagents (fluobodies) that were produced recombinantly in bacteria and are directly suitable for fluorescence microscopy and flow cytometry. Immune-detection identified also cysts and anti-Alexandrium fluobodies showed no cross-reactivity with indigenous not-toxic phytoplankton microalgae belonging to different geni. The fluobodies were able to bind selectively to the target cells in both fixed and fresh samples with minimal processing.

The aim of this work was to test whether salinity is the driving factor for size and physiological differences in the dinoflagellate Prorocentrum cordatum. Three strains, isolated from 3 sites with different salinities (the central Baltic Sea, Sweden, salinity 8; Chesapeake Bay, Maryland, USA, salinity 16; and the Gulf of Trieste, northern Adriatic Sea, Italy, salinity 32), were included in this study. Following their molecular and morphological characterisation, specific growth rates and dimensions were examined for each strain cultivated at all 3 salinities (i.e. 8, 16 and 32). The Adriatic strain always showed the largest cells and the Baltic strain the smallest. Adriatic and American strains showed lower specific growth rates at salinity 8 and highest at salinity 16. The Baltic strain growth rates were similar at all salinities. As salinity 16 proved to be optimal for the strains, additional parameters were further compared at this salinity: cell volume, particulate organic carbon, particulate nitrogen, delayed fluorescence and in vivo chl a fluorescence. The Adriatic strain had the highest particulate organic carbon, particulate nitrogen, and delayed and in vivo fluorescence. Conversely, the Baltic strain had the lowest values. Although the strains had different specific growth rates with respect to salinity, this was not the main driving factor for the different sizes and photosynthetic activity among the strains. Thus, although there was low genetic variability among strains from the 3 geographical areas, intraspecific variability was suggested.

Pseudo-nitzschia H. Peragallo (1900) is a globally distributed genus of pennate diatoms that are important components of phytoplankton communities worldwide. Some members of the genus produce the neurotoxin domoic acid, so regular monitoring is in place. However, the identification of toxic members in routine samplings remains problematic. In this study, the diversity and seasonal occurrence of Pseudo-nitzschia species were investigated in the Gulf of Trieste, a shallow gulf in the northern Adriatic Sea. We used time series data from 2005 to 2018 to describe the seasonal and inter-annual occurrence of the genus in the area and its contribution to the phytoplankton community. On average, the genus accounted for about 15 % of total diatom abundance and peaked in spring and autumn, with occasional outbreaks during summer and large inter-annual fluctuations. Increased water temperature and decreased salinity positively affected the presence of some members of the genus, while strong effects could be masked by an unsuitable definition of the species complexes used for monitoring purposes. Therefore, combining morphological (TEM) and molecular analyses by sequencing the ITS, 28S and rbcL markers, eight species were identified from 83 isolated monoclonal strains: P. calliantha, P. fraudulenta, P. delicatissima, P. galaxiae, P. mannii, P. multistriata, P. pungens and P. subfraudulenta. A genetic comparison between the isolated strains and other strains in the Mediterranean was carried out and rbcL was inspected as a potential barcode marker in respect to our results. This is the first study in the Gulf of Trieste on Pseudo-nitzschia time series from a long-term ecological research (LTER) site coupled with molecular data. We show that meaningful ecological conclusions can be drawn by applying integrative methodology, as opposed to the approach that only considers species complexes. The results of this work will provide guidance for further monitoring efforts as well as research activities, including population genetics and genomics, associated with seasonal distribution and toxicity profiles.

Some euglenids, a family of aquatic unicellular organisms, can develop highly concerted, large-amplitude peristaltic body deformations. This remarkable behaviour has been known for centuries. Yet, its function remains controversial, and is even viewed as a functionless ancestral vestige. Here, by examining swimming Euglena gracilis in environments of controlled crowding and geometry, we show that this behaviour is triggered by confinement. Under these conditions, it allows cells to switch from unviable flagellar swimming to a new and highly robust mode of fast crawling, which can deal with extreme geometric confinement and turn both frictional and hydraulic resistance into propulsive forces. To understand how a single cell can control such an adaptable and robust mode of locomotion, we developed a computational model of the motile apparatus of Euglena cells consisting of an active striated cell envelope. Our modelling shows that gait adaptability does not require specific mechanosensitive feedback but instead can be explained by the mechanical self-regulation of an elastic and extended motor system. Our study thus identifies a locomotory function and the operating principles of the adaptable peristaltic body deformation of Euglena cells., Comment: 8 pages, 4 figures

The future growing demand of fossil fuels likely will lead to an increased deployment of liquefied natural gas terminals. However, some concerns exist about their possible effects on the marine environment and biota. Such plants showed to cause the production of foam, as occurred at the still operative terminal of Porto Viro (northern Adriatic Sea). Here, we present results from two microcosm experiments focused on the effects of such foam on microbially mediated degradation processes and its consequent incorporation within the pelagic food web. Such material could be considered as a heterogeneous matrix of both living and non-living organic matter, which constitutes an important substrate for exoenzymes as suggested by the faster hydrolytic rates measured in the treatment microcosms. In the second experiment, a quite immediate and efficient carbon transfer to planktonic biomass through prokaryotic incorporation and consequent predation by heterotrophic flagellates was highlighted. Although no negative effect was evidenced on the overall microbes' growth and foam-derived C seemed to be easily reworked and transferred to higher trophic levels, an important reduction in biodiversity was evidenced for microalgae. Among them, mixotrophic organisms seemed to be favoured suggesting that the addition of foam could cause a modification of the microbial community structure.

Significance Active flagella provide the propulsion mechanism for a large variety of swimming eukaryotic microorganisms, from protists to sperm cells. Planar and helical beating patterns of these structures are recurrent and widely studied. The fast spinning motion of the locomotory flagellum of the alga Euglena gracilis constitutes a remarkable exception to these patterns. We report a quantitative description of the 3D flagellar beating in swimming E. gracilis. Given their complexity, these shapes cannot be directly imaged with current microscopy techniques. We show how to overcome these limitations by developing a method to reconstruct in full the 3D kinematics of the cell from conventional 2D microscopy images, based on the exact characterization of the helical motion of the cell body., The flagellar swimming of euglenids, which are propelled by a single anterior flagellum, is characterized by a generalized helical motion. The 3D nature of this swimming motion, which lacks some of the symmetries enjoyed by more common model systems, and the complex flagellar beating shapes that power it make its quantitative description challenging. In this work, we provide a quantitative, 3D, highly resolved reconstruction of the swimming trajectories and flagellar shapes of specimens of Euglena gracilis. We achieved this task by using high-speed 2D image recordings taken with a conventional inverted microscope combined with a precise characterization of the helical motion of the cell body to lift the 2D data to 3D trajectories. The propulsion mechanism is discussed. Our results constitute a basis for future biophysical research on a relatively unexplored type of eukaryotic flagellar movement.

Elemental concentrations in micro-PIXE (Particle Induced X-ray Emission) maps of elements in biological tissue slices have been determined using auxiliary information on the sample matrix composition from EBS (Elastic Backscattering Spectroscopy) and STIM (Scanning Transmission Ion Microscopy). The thin sample approximation may be used for evaluating micro-PIXE data in cases, where X-ray absorption in the sample can be neglected and the mass of elements in a selected area can be estimated. The resulting sensitivity amounts to an impressive 10−12 g of the selected elements. Two cases are presented as examples. In the first, we determined the total mass of gold nanoparticles internalized by human monocyte-derived dendritic cells (MDDC). In the second, an inventory of the mass of elements in the micro-particulate material adsorbed at the wall of the lorica of the microzooplankton species Tintinnopsis radix has been created.

We present a method for in situ monitoring of phytoplankton composition changes in a marine environment. The method is based on delayed fluorescence excitation spectra analyzed with CHEMTAX software, which is generally used for determination of phytoplankton communities with HPLC pigment data. Delayed fluorescence (DF) is a photosynthetic parameter that can only be measured in living cells. Algal DF excitation spectra are group-specific, based on their composition of photosynthetic pigments. DF excitation spectra of 14 marine algal species from different families were measured with a delayed fluorescence spectrometer. Mixtures were prepared from northern Adriatic algal species representing six taxonomic groups: dinoflagellates (Prorocentrum minimum), diatoms (Skeletonema costatum), cyanobacteria (Synechococcus sp.), prasinophytes (Micromonas sp.), cryptophytes (Teleaulax sp.), and prymnesiophytes (Isochrysis galbana). The DF excitation spectra (DFS) and HPLC pigment compositions of the mixtures were analyzed with CHEMTAX software. The prediction power of DFS–CHEMTAX method was comparable to HPLC–CHEMTAX.

The changes in the plankton biomass structure in relation to nutrient inputs were studied in the Gulf of Venice (Northern Adriatic Sea), an area characterized by a very marked trophic state variability. The investigation was carried out at two stations, in March, May and July 2005 and 2006, considering the whole water column. The size structure (from picoplankton to mesozooplankton) of both autotrophs and heterotrophs was analysed. Signals of diluted waters and nutrient inputs were more marked in 2005 than in 2006. In 2005, the total plankton biomass was almost double (87 ± 37 μg·C·l−1) that in 2006 (44 ± 26 μg·C·l−1). The variations were determined mainly by phytoplankton, with a 70% decrease, and a shift from a community dominated by microphytoplankton (49 ± 12%) in 2005 to one dominated by bacteria (43 ± 11%) in 2006 was observed. The relationship between the heterotrophic (H) and autotrophic (A) biomass indicated a rapid decline of the H/A ratio with increasing phytoplankton biomass. This study, although temporally limited, is consistent with the results reported for other marine environments and it seems to confirm the importance of nutrient inputs in structuring the biomass of plankton community.

Ostreopsis ovata is an epiphytic potentially toxic dinoflagellate. It has a world-wide distribution, normally associated with other epiphytic or benthic dinoflagellates. In tropical seas O. ovata is often associated with the genera Gambierdiscus, Coolia and Prorocentrum, causing cinguatera fish poisoning. Recently, Ostreopsis spp. blooms in the Tyrrhenian and southern Adriatic Sea have been related to human health problems, such as breathing and skin irritation. Here we report the first record of Ostreopsis cfr. ovata in the Northern Adriatic Sea. O. cfr. ovata was isolated from macroalgae in two areas, the Gulf of Trieste (Italy) and close to Rovinj (Croatia). The microalga was identified by scanning electron microscopy and by fluorescence light microscopy. Size range and thecal pore structure were similar to those described for O. cfr. ovata in previous studies. Ostreopsis cfr. ovata was present on all the macroalgae collected, particularly browns and reds. The microalgal association on macroalgae was mostly composed of Ostreopsis sp., Coolia monotis and Coscinodiscus sp.

Delayed luminescence (DL), also termed delayed fluorescence or delayed light emission, is the phenomenon of long-lived light emission by plants and cyanobacteria after being illuminated with light and put into darkness. Culture growth of three Prorocentrum minimum strains was studied with DL measurements. DL decay kinetics was measured from 1‐60 s after a pulse of white light. The strains used were from the Adriatic Sea (PmK), from Chesapeake Bay, USA (D5), and from the Baltic Sea (BAL), cultured at salinity of 32, 16, and 8 (practical salinity scale), respectively. The strains differed in cell size and chlorophyll a content (PmK > D5 > BAL), as well as in DL parameters. The DL results were compared to standard measurements of culture density and carbon content (calculated from biovolumes). DL decay curves had a specific peak, which changed with culture growth and showed more similarities between the strains PmK and D5. The DL intensity increased with cell density and carbon content in a two-stage process, corresponding to the lag and exponential phases of growth. DL intensity was best correlated with carbon content irrespective of strain and is proposed as an estimate of biomass and for differentiating between lag and exponential phases of growth. # 2004 Elsevier B.V. All rights reserved.

A new species of the dinoflagellate genus Alexandrium, A. tamutum sp. nov., is described based on the results of morphological and phylogenetic studies carried out on strains isolated from two sites in the Mediterranean Sea: the Gulf of Trieste (northern Adriatic Sea) and the Gulf of Naples (central Tyrrhenian Sea). Vegetative cells were examined in light and scanning electron microscopy and resting cysts were obtained by crossing strains of opposite mating type. Alexandrium tamutum is a small-sized species, resembling A. minutum in its small size, the rounded-elliptical shape and the morphology of its cyst. The main diagnostic character of the new species is a relatively wide and large 6th precingular plate (6), whereas that of A. minutum is much narrower and smaller. Contrary to A. minutum, A. tamutum strains did not produce PSP toxins. Phylogenies inferred from the nuclear SSU rDNA and the D1/D2 domains of the LSU nuclear rDNA of five strains of A. tamutum and numerous strains of other Alexandrium species showed that A. tamutum strains clustered in a well supported clade, distinct from A. minutum.

Background The Mediterranean mussel Mytilus galloprovincialis is marine bivalve with a relevant commercial importance as well as a key sentinel organism for the biomonitoring of environmental pollution. Here we report the RNA sequencing of the mussel digestive gland, performed with the aim: a) to produce a high quality de novo transcriptome assembly, thus improving the genetic and molecular knowledge of this organism b) to provide an initial assessment of the response to paralytic shellfish poisoning (PSP) on a molecular level, in order to identify possible molecular markers of toxin accumulation. Results The comprehensive de novo assembly and annotation of the transcriptome yielded a collection of 12,079 non-redundant consensus sequences with an average length of 958 bp, with a high percentage of full-length transcripts. The whole-transcriptome gene expression study indicated that the accumulation of paralytic toxins produced by the dinoflagellate Alexandrium minutum over a time span of 5 days scarcely affected gene expression, but the results need further validation with a greater number of biological samples and naturally contaminated specimens. Conclusion The digestive gland reference transcriptome we produced significantly improves the data collected from previous sequencing efforts and provides a basic resource for expanding functional genomics investigations in M. galloprovincialis. Although not conclusive, the results of the RNA-seq gene expression analysis support the classification of mussels as bivalves refractory to paralytic shellfish poisoning and point out that the identification molecular biomarkers of PSP in the digestive gland of this organism is problematic. Electronic supplementary material The online version of this article (doi:10.1186/1756-0500-7-722) contains supplementary material, which is available to authorized users.

Seasonal seawater temperature increases define optimal growth conditions for Dinoflagellate species which can reach high concentrations in water column and also in filter-feeding organisms like Mytilus galloprovincialis. Commonly produced by Dinophysis and Prorocentrum spp., okadaic acid (OA) and its analogues are responsible for the Diarrheic Shellfish Poisoning (DSP) syndrome in humans. Closure of shellfishing grounds is therefore recommended by the EU when DSP toxin levels in shellfish exceed 16 μg OA 100 g(-1) flesh. Despite not being responsible for casualties either in humans or mussels, DSP outbreaks are considered natural events causing health and economic issues due to the frequency of their occurrence. Since gene expression studies offer a wide range of different solutions, we used a mussel cDNA microarray to evaluate gene expression changes in the digestive gland of mussels fed for five weeks with OA-contaminated nutrient. Among the differentially expressed genes we observed a general up-regulation of transcripts coding for stress proteins, proteins involved in cellular synthesis, and a few not annotated proteins. Overall, at the first time point analyzed we identified 58 candidate transcripts for OA-induced stress in mussels, half of which have unknown function. In this paper we present the first gene expression analysis performed on Mediterranean mussels exposed to okadaic acid. The characterization of these transcripts could be useful for the identification of an early physiological response to OA exposure.

Biological systems are unique matter with very complex morphology and highly heterogeneous chemical composition dominated by light elements. Discriminating qualitatively at the sub-micrometer level the lateral distribution of constituent elements, and correlating it to the sub-cellular biological structure, continues to be a challenge. The low-energy X-ray fluorescence microspectroscopy, recently implemented in TwinMic scanning transmission mode, has opened up new opportunities for mapping the distribution of the light elements, complemented by morphology information provided by simultaneous acquisition of absorption and phase contrast images. The important new information that can be obtained in bio-related research domains is demonstrated by two pilot experiments with specimens of interest for marine biology and food science. They demonstrate the potential to yield important insights into the structural and compositional enrichment, distribution and correlation of essential trace elements in the lorica of Tintinnopsis radix , and the lateral distribution of trace nutrients in the seeds of wheat Triticum aestivum .