Antioxidant Bioprospecting in Microalgae: Characterisation of the Potential of Two Marine Heterokonts from Irish Waters.

Microalgae constitute a heterogeneous and diverse range of organisms capable of accumulating bioactive metabolites, making them promising feedstock for applications in the nutraceutical, functional food, animal feed, biofertilisation or biofuel sectors. There has been renewed interest in recent times in natural sources of antioxidants, particularly as health products and preserving agents. Microalgae strains isolated from aquatic habitats in Ireland were successfully brought into culture. The 91 strains were grown phototrophically in nutrient-enriched media to generate biomass, which was harvested and assessed for antioxidant potential. Extracts were screened for antioxidant activity using a modified volumetric Trolox-ABTS assay and the Folin-Ciocalteu method. Two heterokont marine strains of interest were further studied to ascertain variations in antioxidant capacity across different stages of batch culture growth. The antioxidant activity of extracts of bacillariophyte cf. Stauroneis sp. LACW24 and ocrophyte cf. Phaeothamnion sp. LACW34 increased during growth with a maximum being observed during the late stationary or early death phase (2.5- to 8-fold increases between days 20 and 27). Strains LACW24 and LACW34 contained 5.9 and 3.0 mg g-1 (DW) of the xanthophyll fucoxanthin, respectively. Extracts of strains also showed no cytotoxicity towards mouse cell lines. These results highlight the potential of these strains for biomass valorisation and cultivation upscaling and to be further considered as part of ongoing bioprospecting efforts towards identifying novel species to join the relatively narrow range of commercially exploited marine microalgae species.

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.

Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

An assessment of RNA content in Prymnesium parvum, Prymnesium polylepis, cf. Chattonella sp. and Karlodinium veneficum under varying environmental conditions for calibrating an RNA microarray for species detection.

Traditional methods of identification and enumeration can be somewhat ambiguous when identifying phytoplankton that requires electron microscopic examination to verify specific morphological features. Members of the genus Prymnesium (division Haptophyta), members of the Raphidophyceae and naked dinoflagellates are examples of such phytoplankton whose identification can be difficult. One alternative to traditional microscopy-based methods of identification is to use molecular protocols to detect target species. Methods that measure cellular DNA and RNA content can be used to estimate the number of cells present in a sample. This study investigated the variation of RNA yields in Prymnesium parvum, P. polylepis, cf. Chattonella sp. and Karlodinium veneficum cells grown under different light, temperature, salinity and inorganic nutrient conditions. This information was used to calibrate the signal intensity of a variety of oligonucleotide probes spotted onto the microarrays for the detection of toxic algae (MIDTAL), which is being developed to aid national monitoring agencies and to provide a faster means of identifying and quantifying harmful phytoplankton in water column samples.

An evaluation of the applicability of microarrays for monitoring toxic algae in Irish coastal waters.

The applicability of microarrays to monitor harmful algae across a broad range of ecological niches and toxic species responsible for harmful algal events has been one of the key tasks in the EU Seventh Framework Programme (FP7)-funded Microarrays for the Detection of Toxic Algae project. The technique has a strong potential for improving speed and accuracy of the identification of harmful algae and their toxins to assist monitoring programmes. Water samples were collected from a number of coastal sites around Ireland, including several that are used in the Irish National Phytoplankton and Biotoxin Monitoring Programme. Ribosomal RNA was extracted from filtered field samples, labelled with a fluorescent dye, and hybridised to probes spotted in a microarray format on a glass slide. The fluorescent signal intensity of the hybridisation to >120 probes on the chip was analysed and compared with actual field counts. There was a general agreement between cell counts and microarray signal. Results are presented for field samples taken from a range of stations along the Irish coastline known for harmful algal events during the first field trial (July 2009-April 2010).

Co-occurrence of the West European (Gr.III) and North American (Gr.I) ribotypes of Alexandrium tamarense (Dinophyceae) in Shetland, Scotland.

An investigation into the diversity of the dinoflagellate Alexandrium was carried out during August 2007 within two fjordic sea lochs in the Shetland Isles, Scotland. The co-occurrence in the water column of the non-toxic West European (W.E. or Gr.III) and the neurotoxic North American (N.A. or Gr.I) ribotypes of A. tamarense was demonstrated using fluorescent in situ hybridisation. A patch of A. tamarense (W.E.) localised at approximately 10 m depth and extending over 6 km was detected in 'Clift Sound' with concentrations locally reaching approximately 1 x 10(4) cells l(-1). A. tamarense (N.A.) was also observed there but despite the presence of toxins in net haul samples collected locally, concentrations were low and near limits of detection. Alexandrium concentrations were approximately 1.5 x 10(3) cells l(-1) in 'Vaila Sound', where both W.E. and N.A. ribotypes were detected with equal relative abundances in some samples. Given the patchiness of A. tamarense populations and their possible organisation in thin layer structures, better vertical resolution through fine-scale sampling will be necessary for population dynamic studies. Implications for the shellfish industry are substantial since harmful microalgae patches may not be detected during routine monitoring. Moreover, the co-occurrence of morphologically indistinct toxic and non-toxic ribotypes will necessitate implementing molecular methods for their discrimination.