An evaluation of the applicability of microarrays for monitoring toxic algae in irish coastal waters
McCoy, Gary Robert
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Monitoring of toxic phytoplankton is traditionally carried out by light and electron microscopy. However, the use of molecular methods for identification and quantification are now becoming more prevalent. 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 to the novel MIDTAL (MIcroarrays for the Detection of Toxic ALgae) microarray format. Testing of probe specificity, cross reactivity issues, adaptations and optimisation of protocols are all reported upon during subsequent generations of the MIDTAL microarray. The applicability of microarrays to monitor harmful algae across a broad range of ecological niches and toxic species responsible for harmful algal events was tested in numerous locations around the southern and western coastline of Ireland between 2009 and 2011. Ribosomal RNA was extracted from filtered field samples, labelled with a fluorescent dye and hybridised to oligonucleotide 18S and 28S rDNA probes spotted onto a glass slide. The fluorescent signal intensity of the hybridisation to >140 probes on the chip was analysed and compared with light microscopy counts from field samples. A correlation between RNA content and cell number with microarray signal intensities was one of the main focuses of this project whereby numbers of toxic algal cells could be inferred from the microarray signal. Prymnesium parvum, P. (= Chrysochomulina) polylepis, cf. Chattonella sp. and Karlodinium veneficum cells were grown under different stress conditions (light, temperature, salinity and nutrient), to see if RNA content per cell varied between treatments and over time. The study showed that total rRNA does not always positively correlate with growth rate, with no significant change in rRNA content over time when exposed to the majority of environmental stresses applied and that total rRNA content significantly correlated with increasing cell numbers. Calibration curves for Prymnesium spp. with increasing labelled RNA amounts of 1 ng, 5 ng, 25 ng and 100 ng were performed on both the 2nd and 3rd generation microarrays. These calibrations revealed that the detection limit for both P. parvum and P. polylepis required an RNA amount of ¿5 ng which equates to 8,800 and 3,800 cells respectively. The detection limits of cf. Chattonella and K. veneficum species are also reported to be as low as 1 ng, which is equivalent to <200 cf. Chattonella cells and ~250 K. veneficum cells. During a sampling survey carried out in 2011, there was an extensive bloom of Alexandrium minutum in the North Channel of Cork Harbour and Prorocentrum micans in Bell Harbour of Galway Bay. Both A. minutum and P. micans species probes were successfully detected by the MIDTAL microarray and correlated significantly with light microscopy counts. Additionally, the detection of PSP toxins by the multi SPR biosensor, ELISA and HPLC methods during the A. minutum bloom further validated the presence of this species. The microarray can not only detect what species are co-occurring together in one analysis but can also track the progression of HAB events, giving us an in depth insight into the phytoplankton ecology with the potential to be used as an early warning tool. The main aim of this thesis was to demonstrate the potential use of the MIDTAL microarray and multi SPR biosensor to support national monitoring agencies. Data presented in this thesis support the use of the MIDTAL microarray, which is now commercially available through Microbia Environnement (France), to provide a high-throughput method for fast, accurate detection and quantification of the harmful phytoplankton community and their toxins in natural water samples.