Planktonic ecological networks support quantification of changes in ecosystem health and functioning.

Plankton communities are the foundation of marine food webs and have a large effect on the dynamics of entire ecosystems. Changes in physicochemical factors strongly influence planktonic organisms and their turnover rates, making their communities useful for monitoring ecosystem health. We studied and compared the planktonic food webs of Palude della Rosa (Venice Lagoon, Italy) in 2005 and 2007. The food webs were developed using a novel approach based on the Monte Carlo random sampling of parameters within specific and realistic ranges to derive 1000 food webs for July of each year. The consumption flows involving Strombididae, Evadne spp. and Podon spp. were identified as the most important in splitting food webs of the July of the two years. Although functional nodes (FNs) differed both in presence and abundance in July of the two years, the whole system indicators showed very similar results. Sediment resuspension acted as a source of stress for the Venice Lagoon, being the most used resource by consumers while inhibiting primary producers by increasing water turbidity. Primary production in the water column was mainly generated by benthic FNs. Although the system was near an equilibrium point, it tended to increase its resilience at the expense of efficiency due to stress. This study highlights the role of plankton communities, which can serve to assess ecosystem health.

Nanobody-Dependent Detection of Microcystis aeruginosa by ELISA and Thermal Lens Spectrometry.

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.

A Mediterranean Alexandrium taylorii (Dinophyceae) Strain Produces Goniodomin A and Lytic Compounds but Not Paralytic Shellfish Toxins.

Species of the dinophyte genus Alexandrium are widely distributed and are notorious bloom formers and producers of various potent phycotoxins. The species Alexandrium taylorii is known to form recurrent and dense blooms in the Mediterranean, but its toxin production potential is poorly studied. Here we investigated toxin production potential of a Mediterranean A. taylorii clonal strain by combining state-of-the-art screening for various toxins known to be produced within Alexandrium with a sound morphological and molecular designation of the studied strain. As shown by a detailed thecal plate analysis, morphology of the A. taylorii strain AY7T from the Adriatic Sea conformed with the original species description. Moreover, newly obtained Large Subunit (LSU) and Internal Transcribed Spacers (ITS) rDNA sequences perfectly matched with the majority of other Mediterranean A. taylorii strains from the databases. Based on both ion pair chromatography coupled to post-column derivatization and fluorescence detection (LC-FLD) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis it is shown that A. taylorii AY7T does not produce paralytic shellfish toxins (PST) above a detection limit of ca. 1 fg cell-1, and also lacks any traces of spirolides and gymnodimines. The strain caused cell lysis of protistan species due to poorly characterized lytic compounds, with a density of 185 cells mL-1 causing 50% cell lysis of cryptophyte bioassay target cells (EC50). As shown here for the first time A. taylorii AY7T produced goniodomin A (GDA) at a cellular level of 11.7 pg cell-1. This first report of goniodomin (GD) production of A. taylorii supports the close evolutionary relationship of A. taylorii to other identified GD-producing Alexandrium species. As GD have been causatively linked to fish kills, future studies of Mediterranean A. taylorii blooms should include analysis of GD and should draw attention to potential links to fish kills or other environmental damage.

Ecological time series and integrative taxonomy unveil seasonality and diversity of the toxic diatom Pseudo-nitzschia H. Peragallo in the northern Adriatic Sea.

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.

Swimming Euglena respond to confinement with a behavioral change enabling effective crawling.

Some euglenids, a family of aquatic unicellular organisms, can develop highly concerted, large amplitude peristaltic body deformations. This remarkable behavior 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 behavior 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 modeling 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.

In vitro isolation of nanobodies for selective Alexandrium minutum recognition: A model for convenient development of dedicated immuno-reagents to study and diagnostic toxic unicellular algae.

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.

Kinematics of flagellar swimming in Euglena gracilis: Helical trajectories and flagellar shapes.

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.

Foam production as a side effect of an offshore liquefied natural gas terminal: how do plankton deal with it?

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.

RNA sequencing and de novo assembly of the digestive gland transcriptome in Mytilus galloprovincialis fed with toxinogenic and non-toxic strains of Alexandrium 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.

Mediterranean mussel gene expression profile induced by okadaic acid exposure.

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.

Low-energy X-ray fluorescence microscopy opening new opportunities for bio-related research.

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.

First record of Ostreopsis cfr. ovata on macroalgae in the Northern Adriatic Sea.

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.

Pelagostrobilidium neptuni (Montagnes and Taylor, 1994) and Strombidium biarmatum nov. spec. (Ciliophora, Oligotrichea): phylogenetic position inferred from morphology, ontogenesis, and gene sequence data.

Morphological data from life, protargol impregnation, and scanning electron microscopy were combined with genetic data not only to describe the marine plankton ciliates Pelagostrobilidium neptuni (Montagnes and Taylor, 1994) Petz, Song, and Wilbert, 1995 and Strombidium biarmatum nov. spec., but also to elucidate their phylogenetic relationships. Additionally, the ontogenesis of P. neptuni was studied and the diagnosis of the genus Pelagostrobilidium was improved due to further data from the newly affiliated species P. epacrum (Lynn and Montagnes, 1988) nov. comb. (basionym: Strobilidium epacrum Lynn and Montagnes, 1988). The phylogenetic analysis of the small subunit ribosomal RNA genes matched the morphologic and ontogenetic assigning of P. neptuni to the choreotrichid family Strobilidiidae. The considerable genetic distance of d = 0.074 between P. neptuni and Strobilidium caudatum corroborated the morphological differences and thus the maintenance of the genus Pelagostrobilidium. Strombidium biarmatum nov. spec. is a typical member of the genus, except for the two types of extrusomes ("trichites"): ~12 × 0.5 µm, needle-shaped ones attached anterior to the girdle kinety and ~6 × 0.5 µm, rod-shaped ones at the distal end of the intermembranellar ridges. Its flask-shaped resting cysts have several strong spines. In accordance with the morphologic data, S. biarmatum is placed within the order Oligotrichida by gene sequence analysis. The great genetic distances within the oligotrichids support the diversity found in morphologic and ontogenetic studies.

Morphologic and genetic variability in the marine planktonic ciliate Laboea strobila Lohmann, 1908 (Ciliophora, Oligotrichia), with notes on its ontogenesis.

Laboea strobila Lohmann, 1908 is a conspicuous oligotrich ciliate in the marine plankton. In order to compare different populations, the morphology of specimens from the Mediterranean Sea, North Sea, and Irish Sea was investigated using live observation, protargol impregnation, and scanning electron microscopy. Furthermore, the PCR-amplified products of the SSrRNA gene from a monoclonal culture of L. strobila from the Mediterranean Sea were sequenced and aligned with sequences of other oligotrichs, including a population of L. strobila from the Atlantic coast of the USA. Finally, the data from the ecological literature were summarized and the cultivation methods were described. The SSrRNA gene sequences of the two distantly located L. strobila populations from the North Atlantic are identical. Likewise, the morphometrics of most populations so far investigated after protargol impregnation (i.e. from the North Atlantic) do not show obvious differences. In all computed phylogenetic trees, L. strobila groups with Strombidium species, forming a monophyletic taxon corresponding to the subclass Oligotrichia. These results are corroborated by the ontogenetic comparison. Since no type species was fixed for Laboea Lohmann, 1908, L. strobila was designated in the present paper.