Nicolas Touzet

Characterization of Nontoxic and Toxin-Producing Strains of Alexandrium minutum (Dinophyceae) in Irish Coastal Waters

ABSTRACT A comparative analysis of the morphology, toxin composition, and ribosomal DNA (rDNA) sequences was performed on a suite of clonal cultures of the potentially toxic dinoflagellate Alexandrium minutum Halim. These were established from resting cysts or vegetative cells isolated from sediment and water samples taken from the south and west coasts of Ireland. Results revealed that strains were indistinguishable, both morphologically and through the sequencing of the D1-D2 domain of the large subunit and the ITS1-5.8S-ITS2 regions of the rDNA. High-performance liquid chromatography fluorescence detection analysis, however, showed that only strains derived from retentive inlets on the southern Irish coast synthesized paralytic shellfish poisoning (PSP) toxins (GTX2 and GTX3), whereas all strains of A. minutum isolated from the west coast were nontoxic. Toxin analysis of net hauls, taken when A. minutum vegetative cells were in the water column, revealed no PSP toxins in samples from Killary Harbor (western coast), whereas GTX2 and GTX3 were detected in samples from Cork Harbor (southern coast). These results confirm the identity of A. minutum as the most probable causative organism for historical occurrences of contamination of shellfish with PSP toxins in Cork Harbor. Finally, random amplification of polymorphic DNA was carried out to determine the degree of polymorphism among strains. The analysis showed that all toxic strains from Cork Harbor clustered together and that a separate cluster grouped all nontoxic strains from the western coast.

Evaluation of taxa-specific real-time PCR, whole-cell FISH and morphotaxonomy analyses for the detection and quantification of the toxic microalgae Alexandrium minutum (Dinophyceae), Global Clade ribotype.

The dinoflagellate genus Alexandrium contains neurotoxin-producing species that have adversely affected the aquaculture industry in many countries. The morphological similarity between Alexandrium species has led to the development of molecular methods for the discrimination, enumeration and monitoring of toxic and nontoxic species. A quantitative real-time PCR assay (qRT-PCR) targeting the internal transcribed spacer 1-5.8S rRNA gene using hybridization probe technology was developed for the potentially toxic species Alexandrium minutum (Global Clade) (GC). The assay was specific with a detection limit of less than one cell equivalent. The assay was used to detect and quantify A. minutum (GC) in seawater samples collected during summer 2007 in Cork Harbour, Ireland. The results were compared with those obtained using whole-cell FISH (WC-FISH) and morphotaxonomy analyses. Alexandrium minutum did not reach high bloom concentrations over the sampling period (maximum of c. 6 x 10(4) cells L(-1)), and the average concentrations determined using qRT-PCR, WC-FISH and morphotaxonomy did not significantly differ in eight of nine comparisons. Regression curves showed positive relationships between the methods; WC-FISH and qRT-PCR slightly under- and overestimated, respectively, the A. minutum concentrations compared with the morphotaxonomy method. The qRT-PCR assay for A. minutum (GC) offers high-throughput sample analysis and may prove suitable for implementation in microalgae monitoring programmes and assist in population dynamics studies of the species.

Discrimination of Alexandrium andersoni and A. minutum (Dinophyceae) using LSU rRNA-targeted oligonucleotide probes and fluorescent whole-cell hybridization

N. Touzet and R. Raine. 2007. Discrimination of Alexandrium andersoni and A. minutum (Dinophyceae) using LSU rRNA-targeted oligonucleotide probes and fluorescent whole-cell hybridization. Phycologia 46: 168–177. DOI: 10.2216/06-11.1 Toxic marine dinoflagellates from the genus Alexandrium have been responsible for paralytic shellfish poisoning (PSP) throughout the world. Their monitoring relies on spatial and temporal sampling strategies and requires the reliable identification and enumeration of vegetative stages in order to enable the development of early warning policies. The accurate discrimination between Alexandrium species is labour intensive and requires taxonomic expertise as the genus contains morphologically similar toxic and non-toxic species. In Ireland, PSP outbreaks so far have been limited to Cork Harbour, a retentive inlet located on the south coast of the country, where the causative organism has been identified as A. minutum. Recently, the non-toxic and morphologically similar species A. andersoni has been detected on the south west coast of Ireland. In routine monitoring, Alexandrium spp. are identified on the basis of morphological features by conventional light microscopy, a method which does not allow their characterization at the species level. The development and application of large subunit (LSU) rRNA-targeted oligonucleotide probes for the detection and quantification of A. minutum (Global clade) and A. andersoni by whole-cell fluorescent in situ hybridization (FISH) is reported. The specificity and sensitivity of the two probe sets (MinA and AndA′ +C) were evaluated against Alexandrium species, including A. tamarense, A. tamutum and A. ostenfeldi, and a range of common dinoflagellates usually co-occurring with Alexandrium in Irish coastal waters. No cross-reactivity was observed with any of the strains tested or with phytoplankton species present in field samples rich in dinoflagellates. The format of the assay overcame possible matrix effects, such as probe adsorption, and allowed the reliable labelling of at least 1000 cells. Furthermore, the simultaneous use of calcofluor during the assays permitted the confirmation of the probe diagnostics by examining the general plate structure of the thecae of labelled cells.

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).

Molecular fingerprinting of lacustrian cyanobacterial communities: regional patterns in summer diversity

The assessment of lacustrian water quality is necessary to comply with environmental regulations. At the regional scale, difficulties reside in the selection of representative lakes. Given the risks towards water quality associated with phytoplankton blooms, a mesoscale survey was carried out in Irish lakes to identify patterns in the distribution and diversity of planktonic cyanobacteria. A stratified sampling strategy was carried out via geographic information systems (GIS) analysis of river catchment attributes due to the range of hydrogeomorphological features and the high number of lakes within the study area. 16S rRNA gene denaturing gradient gel electrophoresis analysis showed variation between the cyanobacterial communities sampled, with lower occurrence of cyanobacteria in August concomitant to increased wind and precipitation regimes. Multivariate analysis delineated three ecoregions based on land cover typology and revealed significant patterns in the distribution of cyanobacterial diversity. A majority of filamentous cyanobacteria genotypes occurred in larger lakes contained river catchments with substantial forest cover. In contrast, higher diversity of spherical cyanobacteria genotypes was observed in lakes of lesser trophic state. In the context of aquatic resource management, the combined use of GIS-based sampling strategy and molecular methods offers promising prospects for assessing microbial community structure at varying scales of space and time.

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.

Phycoremediation of landfill leachate with the chlorophyte Chlamydomonas sp. SW15aRL and evaluation of toxicity pre and post treatment

Landfill leachate treatment is an ongoing challenge in the wastewater management of existing sanitary landfill sites due to the complex nature of leachates and their heavy pollutant load. There is a continuous interest in treatment biotechnologies with expected added benefits for resource recovery; microalgal bioremediation is seen as promising in this regard. Toxicity reduction of landfill leachate subsequent to phycoremediation was investigated in this study. The treatment eventuated from the growth of the ammonia tolerant microalgal strain Chlamydomonas sp. SW15aRL using a N:P ratio adjustment in diluted leachate for facilitating the process. Toxicity tests ranging over a number of trophic levels were applied, including bacterial-yeast (MARA), protistean (microalgae growth inhibition test), crustacean (daphnia, rotifer) and higher plant (monocot, dicot) assays. Ammonia nitrogen in the diluted landfill leachate containing up to 158mgl-1 NH4+-N (60% dilution of the original) was reduced by 83% during the microalgal treatment. Testing prior to remediation indicated the highest toxicity in the crustacean assays Daphnia magna and Brachionus calyciflorus with EC50s at 24h of ~ 35% and 40% leachate dilution, respectively. A major reduction in toxicity was achieved with both bioassays post microalgal treatment with effects well below the EC20s. The microalgae inhibition test on the other hand indicated increased stimulation of growth after treatment as a result of toxicity reduction but also the presence of residual nutrients. Several concurrent processes of both biotic and abiotic natures contributed to pollutant reduction during the treatment. Modifying phosphate dosage especially seems to require further attention. As a by-product of the remediation process, up to 1.2gl-1 of microalgal biomass was obtained with ~ 18% DW lipid content.

Comparative summer dynamics of surface cyanobacterial communities in two connected lakes from the west of Ireland.

The eutrophication of lakes is typically associated with high biomass proliferations of potentially toxic cyanobacteria. At a regional level, the sustainable management of water resources necessitates an approach that recognises the interconnectivity of multiple water systems within river catchments. This study examined the dynamics in summer diversity of planktonic cyanobacterial communities and microcystin toxin concentrations in two inter-connected lakes from the west of Ireland prone to nutrient enrichment. DGGE analysis of 16S rRNA gene amplicons of genotype-I cyanobacteria (typically spherical) showed changes in the communities of both Lough Corrib and Ballyquirke Lough throughout the summer, and identified cyanobacterial genotypes both unique and shared to both lakes. Microcystin concentrations, estimated via the protein phosphatase 2A inhibition assay, were greater in August than in July and June in both lakes. This was concomitant to the increased occurrence of Microcystis as evidenced by DGGE band excision and subsequent sequencing and BLAST analysis. RFLP analysis of PCR amplified mcy-A/E genes clustered together the August samples of both lakes, highlighting a potential change in microcystin producers across the two lakes. Finally, the multiple factor analysis of the combined environmental data set for the two lakes highlighted the expected pattern opposing greater water temperature and chlorophyll concentration against macronutrient concentrations, but also indicated a negative relationship between microcystin concentration and cyanobacterial diversity, possibly underlining allelopathic interactions. Despite some element of connectivity, the dissimilarity in the composition of the cyanobacterial assemblages and the timing of community change in the two lakes likely were a reflexion of niche differences determined by meteorologically-forced variation in physico-chemical parameters in the two water bodies.