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Organisms adaptable to extreme conditions share the ability to establish protective biofilms or secrete defence toxins. The extracellular substances that are secreted may contain monosaccharides and other toxic compounds, but environmental conditions influence biofilm characteristics. Microorganisms that are present in the same environment achieve similar compositions, regardless of their phylogenetic relationships. Alternatively, cyanobacteria phylogenetically related may live in different environments, but we ignore if their physiological answers may be similar. To test this hypothesis, two strains of cyanobacteria that were both ascribed to the genus Halomicronema were isolated. H. metazoicum was isolated in marine waters off the island of Ischia (Bay of Naples, Italy), free living on leaves of Posidonia oceanica. Halomicronema sp. was isolated in adjacent thermal waters. Thus, two congeneric species adapted to different environments but diffused in the same area were polyphasically characterized by microscopy, molecular, and toxicity analyses. A variable pattern of toxicity was exhibited, in accordance with the constraints imposed by the host environments. Cyanobacteria adapted to extreme environments of thermal waters face a few competitors and exhibit a low toxicity; in contrast, congeneric strains that have adapted to stable and complex environments as seagrass meadows compete with several organisms for space and resources, and they produce toxic compounds that are constitutively secreted in the surrounding waters., Competing Interests: The authors declare no conflict of interest. The funding sponsors and institutions had no role in the design of the study, in the collection, analyses or interpretation of the data as well as in the writing of the manuscript and in the decision to publish results.

Diatoms are unicellular eukaryotic organisms that play a key ecological and biogeochemical role in oceans as major primary producers. Recently, these microalgae have also attracted interest as a promising source of functional products with widespread relevance. Progress in the knowledge of cell and molecular biology of diatoms is envisaged as a key step to understanding regulation of their life cycle in marine environments as well as facilitating their full and profitable exploitation by biotechnological platforms. Recently, we identified sterol sulfates (StS) as regulatory molecules of cell death in the diatom Skeletonema marinoi . As these compounds may have a general role in diatom physiology and chemical signals in aquatic systems, we investigated a suitable tool for their analysis in laboratory and field samples. Herein, we describe a sensitive, fast, and efficient ultra performance liquid chromatography⁻mass spectrometry (UPLC⁻MS) method for qualitative and quantitative analysis of StS from crude extract of diatoms and other microalgae. The method was applied to 13 different strains of our collection of marine protists. This first study suggested a species-specific distribution of StS and identified the sulfated derivatives of 24-methylene cholesterol and 24-methyl cholesterol as the most common members in diatoms.

Cyanobacteria contribute to the ecology of various marine environments, also for their symbioses, since some of them are common hosts of sponges and ascidians. They are also emerging as an important source of novel bioactive secondary metabolites in pharmacological (as anticancer drugs) and biotechnological applications. In the present work we isolated a cyanobacteria in a free-living state from leaves of the seagrass Posidonia oceanica leaves. This newly collected strain was then cultivated under two laboratory conditions, and then characterized by combining morphological observation and molecular studies based on 16S rRNA gene sequences analysis. The strain showed 99% pairwise sequence identity with Halomicronema metazoicum ITAC101, never isolated before as a free-living organisms, but firstly described as an endosymbiont of the Mediterranean marine spongae Petrosia ficiformis, under the form of a filamentous strain. Further studies will investigate the actual role of this cyanobacterium in the leaf stratum of P. oceanica leaves, given its demonstrated ability to influence the vitality and the life cycle of other organisms. In fact, its newly demonstrated free-living stage, described in this study, indicate that Phormidium-like cyanobacteria could play important roles in the ecology of benthic and planktonic communities., Competing Interests: The authors have declared that no competing interests exist.

Diatoms are unicellular algae playing a key role as photosynthetic organisms in the world's ocean food webs. The chemical ecology of planktonic diatoms is well documented, but few studies have reported on the effects of benthic diatoms on their consumers, also due to difficulties in the collection, quantification and massive culturing of benthic species. Here for the first time we investigate the effects of feeding on two abundantly occurring benthic diatoms, Nanofrustulum shiloi and Cylindrotheca closterium, isolated from the leaves of the seagrass Posidonia oceanica, on the sea urchin Paracentrotus lividus. Adult P. lividus were fed for one month on diets of either one of the two diatoms and on the green alga Ulva rigida, used as a feeding control. By combining morphological, metabolomic and de novo transcriptomic approaches, we demonstrate toxigenic effect on embryos generated by females fed with these benthic diatoms. Furthermore, chemical analysis reveal the presence of polyunsaturated aldehydes only for N. shiloi, and a high production of other oxylipins (cytotoxic compounds on their grazers and on cancer cell lines) for both diatoms, including some additional peaks not correlated to the canonic oxylipins commonly observed in planktonic diatoms. These findings open new perspectives in the study of diatom secondary metabolites influencing their grazers.

Sex reversal is a process observed in several marine organisms, including some lineages of caridean shrimps. We investigated the gonopore shape and size, to study the sex reversal using Hippolyte inermis as a model. A method was developed which can be applied to identify the sex in juveniles of H. inermis , especially, useful when the standard method of sex assessment is not applicable. The position and the shape of gonopores was recorded under a light macroscope. The sex of mature individuals was then determined by observing the presence/absence of the appendix masculina . In addition, analysis of ontogenetic changes of gonopores were performed to compare their morphology with other species of shrimps whose gonopore morphology was previously known. Female gonopores are located at the far proximo-medial end of the third pair of pereiopod coxae and distally they bear cup-shaped structures, whilst male gonopores are located at the far proximo-medial end of the fifth pair of pereiopod coxae and they have a simpler structure. The shape and structure of gonopores in H. inermis resembled that of other caridean decapods. Intersex individuals were never observed, although this species was demonstrated to be protandric. This observation confirmed previous assumptions indicating that the process of sex reversal is very fast in H. inermis and that it takes place within a single moult. The identification of sex based on the position and shape of gonopores is feasible in this species, and it provides helpful insights for studying sex reversal in small decapods.

The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism.

The abundance and the distribution of trophic resources available for consumers influence the productivity and the diversity of natural communities. Nevertheless, assessment of the actual abundance of food items available for individual trophic groups has been constrained by differences in methods and metrics used by various authors. Here we develop an index of food abundance, the framework of which can be adapted for different ecosystems. The relative available food index (RAFI) is computed by considering standard resource conditions of a habitat and the influence of various generalized anthropogenic and natural factors. RAFI was developed using published literature on food abundance and validated by comparison of predictions versus observed trophic resources across various marine sites. RAFI tables here proposed can be applied to a range of marine ecosystems for predictions of the potential abundance of food available for each trophic group, hence permitting exploration of ecological theories by focusing on the deviation from the observed to the expected.

Several plants and invertebrates interact and communicate by means of volatile organic compounds (VOCs). These compounds may play the role of infochemicals, being able to carry complex information to selected species, thus mediating inter- or intra-specific communications. Volatile organic compounds derived from the wounding of marine diatoms, for example, carry information for several benthic and planktonic invertebrates. Although the ecological importance of VOCs has been demonstrated, both in terrestrial plants and in marine microalgae, their role as infochemicals has not been demonstrated in seagrasses. In addition, benthic communities, even the most complex and resilient, as those associated to seagrass meadows, are affected by ocean acidification at various levels. Therefore, the acidification of oceans could produce interference in the way seagrass-associated invertebrates recognize and choose their specific environments. We simulated the wounding of Posidonia oceanica leaves collected at two sites (a control site at normal pH, and a naturally acidified site) off the Island of Ischia (Gulf of Naples, Italy). We extracted the VOCs and tested a set of 13 species of associated invertebrates for their specific chemotactic responses in order to determine if: a) seagrasses produce VOCs playing the role of infochemicals, and b) their effects can be altered by seawater pH. Our results indicate that several invertebrates recognize the odor of wounded P. oceanica leaves, especially those strictly associated to the leaf stratum of the seagrass. Their chemotactic reactions may be modulated by the seawater pH, thus impairing the chemical communications in seagrass-associated communities in acidified conditions. In fact, 54% of the tested species exhibited a changed behavioral response in acidified waters (pH 7.7). Furthermore, the differences observed in the abundance of invertebrates, in natural vs. acidified field conditions, are in agreement with these behavioral changes. Therefore, leaf-produced infochemicals may influence the structure of P. oceanica epifaunal communities, and their effects can be regulated by seawater acidification.

Benthic diatoms of the genus Cocconeis contain a specific apoptogenic activity. It triggers a fast destruction of the androgenic gland in the early post-larval life of the marine shrimp Hippolyte inermis, leading to the generation of small females. Previous in vitro investigations demonstrated that crude extracts of these diatoms specifically activate a dose-dependent apoptotic process in human cancer cells (BT20 breast carcinoma) but not in human normal lymphocytes. Here, a bioassay-guided fractionation has been performed to detect the apoptogenic compound(s). Various HPLC separation systems were needed to isolate the active fractions, since the apoptogenic metabolite is highly active, present in low amounts and is masked by abundant but non-active cellular compounds. The activity is due to at least two compounds characterized by different polarities, a hydrophilic and a lipophilic fraction. We purified the lipophilic fraction, which led to the characterization of an active sub-fraction containing a highly lipophilic compound, whose molecular structure has not yet been identified, but is under investigation. The results point to the possible medical uses of the active compound. Once the molecular structure has been identified, the study and modulation of apoptotic processes in various types of cells will be possible.

Context: The marine diatoms Cocconeis scutellum Ehrenberg (Bacillariophyceae) are known to trigger apoptosis in the androgenic gland of the Mediterranean crustacean Hippolyte inermis Leach (Decapoda), affecting the shrimp's sex reversal., Objective: The aim of this study was to evaluate a possible apoptotic effect of extracts and fractions from these microalgae also on human tissues., Materials and Methods: The chemical profile of C. scutellum was determined by gas chromatography-mass spectrometry (GC-MS) and, afterwards, organic extracts and fractions from the diatoms were used to treat to breast cancer BT20 cells. Double labeling with annexin V-FITC and isotonic propidium iodide (PI) along with flow cytometry analysis enabled the evaluate of cell apoptosis and viability, whereas hypotonic PI staining was used to analyze the cell cycle in BT20 lines. The involvement of specific caspases was studied by Western blotting., Results: Results demonstrated that the diethyl ether extract and, in particular, fraction 3, the richest fraction in eicosapentaenoic acid (EPA) from the diethyl ether extract, selectively induced apoptosis (up to 89.2% at 1 μg/well of fraction 3) and decreased viability in BT20 cells. The apoptotic effect was displayed in a concentration and time-dependent manner, by activating caspases-8 and 3, and arresting the progression of the cell cycle from S to G2-M phase. EPA alone showed similar apoptotic effects in BT20 cells., Discussion and Conclusion: The study demonstrates the apoptotic activity of C. scutellum diatoms on breast cancer cells and suggests their potential use as a source of apoptotic compounds.

The optimal conditions for the growth of two conspecific benthic diatoms were defined through factorial experimentation. We investigated the roles of light spectrum, nutrient availability, and culture conditions on the laboratory production of Cocconeis scutellum scutellum Ehrenb. and C. scutellum parva Grunow. Diatoms were cultivated in petri dishes, and inverted optical microscopy was used to periodically record their abundance. Growth curves were constructed from these data for each culture condition. In addition, at the end of the experiment we performed weight measurements to determine the total production for each of the considered conditions. We found that cultivation in nonsealed (NS) petri dishes (permitting gas exchange) represented the most productive technique. Cell density and biomass varied among light spectra, although this effect was inconsistent. For example, the Sylvania Gro-Lux lamp (GL) produced the lowest cell density but highest biomass, suggesting that it may promote the production of larger cells. Surprisingly, of the culture media tested, f/2 (a media commonly used for the culture of diatoms) was the least productive. Diatom density and biomass were variably dependent on the combination of experimental culture conditions and strain used. These physical and chemical factors act mainly on given features of the diatom growth curve. These results permitted us to devise adequate culture protocols, to produce a biotechnologically important substance: a proapoptotic compound that specifically destroys the androgenic gland of a shrimp and could find novel applications in human medicine., (© 2011 Phycological Society of America.)

Confocal Laser Scanning Microscope techniques have been applied to study the developmental biology of marine copepods and decapod larvae. The lipophylic probes DiI and DiOC(6) were used to study both the external and internal morphology of these crustaceans, whereas the same DiOC(6) and the specific nuclear probe Hoechst 33342 were used to study embryonic development of copepods in vivo. To distinguish viable from non-viable copepod embryos, the vital dye dichlorodihydrofluorescein diacetate (H(2)DCFDA) was used. Major advantages and difficulties in the use of these non-invasive techniques in studies of the reproductive biology of marine crustaceans are discussed., (Copyright 2003 Wiley-Liss, Inc.)

This was a random double-blind study versus placebo designed to assess the effectiveness and tolerability of defibrotide in patients with chronic peripheral vascular disease (PVD), Leriche stage II. A total of 20 male outpatients (mean age 58.8 years) were selected and treated with placebo for 2 weeks (run-in period), and then allocated randomly to treatment with defibrotide (200 mg/day, intramuscular, n = 10) or matched placebo (n = 10) for 90 days. Before intake and at 15, 30, 60, and 90 days of treatment, each patient received a Doppler ultrasound examination of the lower limbs, and the residual perfusion index (Winsor) was calculated at rest and immediately after a symptom-limited treadmill test. Absolute walking distance was measured in addition. Blood samples were taken for routine laboratory tests before intake and at 30 and 90 days of treatment. All patients completed the trial without adverse effects; no meaningful alterations of laboratory test returns were detected. Patients receiving defibrotide showed a significant increase of the Winsor index after exercise at 90 days versus basal (mean 0.47 +/- 0.04 vs. 0.43 +/- 0.05, p less than 0.05), and a parallel increase of walking distance (basal 288 +/- 42.3 m vs. 368 +/- 46.6 at 60 days, p less than 0.05, and 407 +/- 64.3 m at 90 days, p less than 0.01). No improvement was seen in the placebo group. These preliminary results suggest that defibrotide may prove beneficial to patients with PVD; further studies are needed to find the most appropriate dosage regime.