Laura Escalera

Bioactivity Screening of Microalgae for Antioxidant, Anti-Inflammatory, Anticancer, Anti-Diabetes, and Antibacterial Activities

Marine microalgae are considered a potentially new and valuable source of biologically active molecules for applications in the food industry as well as in the pharmaceutical, nutraceutical and cosmetic sectors. They can be easily cultured, have short generation times and enable an environmentally-friendly approach to drug discovery by overcoming problems associated with the over-utilization of marine resources and the use of destructive collection practices. In this study, 21 diatoms, 7 dinoflagellates and 4 flagellate species were grown in three different culturing conditions and the corresponding extracts were tested for possible antioxidant, anti-inflammatory, anticancer, anti-diabetes, antibacterial and anti-biofilm activities. In addition, for two diatoms we also tested two different clones to disclose diversity in clone bioactivity. Six diatom species displayed specific anti-inflammatory, anticancer (blocking human melanoma cell proliferation) and anti-biofilm (against the bacteria Staphylococcus epidermidis) activities whereas, none of the other microalgae were bioactive against the conditions tested for. Furthermore, none of the 6 diatom species tested were toxic on normal human cells. Culturing conditions (i.e. nutrient starvation conditions) greatly influenced bioactivity of the majority of the clones/species tested. This study denotes the potential of diatoms as sources of promising bioactives for the treatment of human pathologies.

PLANOZYGOTE DIVISION AND OTHER OBSERVATIONS ON THE SEXUAL CYCLE OF SEVERAL SPECIES OF DINOPHYSIS (DINOPHYCEAE, DINOPHYSIALES)1

A life‐cycle model has been proposed for Dinophysis, but several transitions between stages of this cycle needed more detailed description. In this study, the steps from mating gamete pairs, cell fusion, nuclear fusion, and the fate of planozygotes were tracked and described from incubations of different sexual‐cycle stages of D. acuminata Clap. et J. Lachm., D. cf. ovum F. Schütt, and D. acuta Ehrenb. There were several pathways for depauperating division and formation of small and intermediate cells; observed mating tubes that connect mating gamete pairs were more delicate than the feeding tube described in D. acuminata; nuclear fusion occurs following cell fusion. Planozygotes were able to divide and produce several vegetative cells 2–3 weeks after incubation. New pathways were added to the revised sexual life‐cycle model of Dinophysis spp. It is hypothesized that planozygotes are the main diploid sexual stage that may be involved in overwintering and seeding strategies. The importance of planozygote division, without further maturation into a resting cyst, as an adaptive strategy for holoplanktonic organisms is discussed.

Molecular probes and microarrays for the detection of toxic algae in the genera Dinophysis and Phalacroma (Dinophyta)

Dinophysis and Phalacroma species containing diarrheic shellfish toxins and pectenotoxins occur in coastal temperate waters all year round and prevent the harvesting of mussels during several months each year in regions in Europe, Chile, Japan, and New Zealand. Toxicity varies among morphologically similar species, and a precise identification is needed for early warning systems. Molecular techniques using ribosomal DNA sequences offer a means to identify and detect precisely the potentially toxic species. We designed molecular probes targeting the 18S rDNA at the family and genus levels for Dinophysis and Phalacroma and at the species level for Dinophysis acuminata, Dinophysis acuta, and Dinophysis norvegica, the most commonly occurring, potentially toxic species of these genera in Western European waters. Dot blot hybridizations with polymerase chain reaction (PCR)-amplified rDNA from 17 microalgae were used to demonstrate probe specificity. The probes were modified along with other published fluorescence in situ hybridization and PCR probes and tested for a microarray platform within the MIDTAL project (http://www.midtal.com). The microarray was applied to field samples from Norway and Spain and compared to microscopic cell counts. These probes may be useful for early warning systems and monitoring and can also be used in population dynamic studies to distinguish species and life cycle stages, such as cysts, and their distribution in time and space.

Ultrastructural features of the benthic dinoflagellate Ostreopsis cf. ovata (Dinophyceae).

The toxic benthic dinoflagellate Ostreopsis cf. ovata has considerably expanded its distribution range in the last decade, posing risks to human health. Several aspects of this species are still poorly known. We studied ultrastructural features of cultivated and natural populations of Ostreopsis cf. ovata from the Gulf of Naples (Mediterranean Sea) using confocal laser scanning, and scanning and transmission electron microscopy. New information on the morphology and location of several sulcal plates was gained and a new plate designation is suggested that better fits the one applied to other Gonyaulacales. The microtubular component of the cytoskeleton, revealed using an anti-β-tubulin antibody, consisted of a cortical layer of microtubules arranged asymmetrically in the episome and in the hyposome, complemented by a complex inner microtubular system running from the sulcal area towards the internal part of the cell. The conspicuous canal was delimited by two thick, burin-shaped lobes ending in a tubular ventral opening. The canal was surrounded by mucocysts discharging their content into it. A similar structure has been reported in other benthic and planktonic dinoflagellates and may be interpreted as an example of convergent evolution in species producing large amounts of mucus.

Cyanobacterial endosymbionts in the benthic dinoflagellate Sinophysis canaliculata (Dinophysiales, Dinophyceae).

Photosynthetic dinoflagellates possess a great diversity of plastids that have been acquired through successful serial endosymbiosis. The peridinin-containing plastid in dinoflagellates is canonical, but many other types are known within this group. Within the Dinophysiales, several species of Dinophysis contain plastids, derived from cryptophytes or haptophytes. In this work, the presence of numerous intracellular cyanobacteria-like microorganisms compartmentalized by a separate membrane is reported for the first time within the benthic dinophysoid dinoflagellate Sinophysis canaliculata Quod et al., a species from a genus morphologically close to Dinophysis. Although the contribution of these cyanobacterial endosymbionts to S. canaliculata is still unknown, this finding suggests a possible undergoing primary endosymbiosis in a dinoflagellate.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n=256) from European waters, collected 2009-2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2×2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Gonyaulax hyalina and Gonyaulax fragilis (Dinoflagellata), two names associated with ‘mare sporco', indicate the same species

Abstract: Aggregates of mucilaginous material have been at times observed in coastal and oceanic sea waters. In the Adriatic Sea (Mediterranean Sea), these events have been known since the 18th century as ‘mare sporco’ (= dirty sea), and in recent years, they have been associated with the dinoflagellate Gonyaulax fragilis; whereas, in New Zealand mucilage events were linked to Gonyaulax hyalina. In summer 2012, during a ‘mare sporco’ event in the Gulf of Naples (Mediterranean Sea), numerous cells of a species tentatively identified as Gonyaulax hyalina were detected in the mucilaginous aggregates. Since the boundary between G. fragilis and G. hyalina was not clear, we compared a strain isolated from the Gulf of Naples in 2012 with two strains from the Adriatic Sea and New Zealand waters, the latter two identified as G. fragilis and G. hyalina, respectively. Detailed light microscopy and scanning electron microscopy observations of the three strains showed that all shared the same thecal plate pattern and morphological features. Partial small subunit and large subunit rDNA regions of the three strains were identical. Based on these results and on a revision of the taxonomic history of the two taxa, we propose that G. fragilis and G. hyalina are the same species. Gonyaulax fragilis was described as Steiniella fragilis by Schütt in 1895 and transferred to G. fragilis by Kofoid in 1911. Gonyaulax hyalina was described a few years later by Ostenfeld and Schmidt in 1901. Thus, the name G. fragilis has priority, while G. hyalina is to be considered a synonym.