Investigating the distribution, seasonal dynamics and toxicity of Azadinium spinosum in Scottish waters using qPCR
The small dinoflagellate Azadinium spinosum produces azaspiracid (AZA) toxins which can contaminate filter feeding shellfish to dangerous levels. Toxin-contaminated shellfish flesh, when consumed by humans, can cause acute intense illness and chronic health issues. Shellfish biotoxins are monitored in Scottish shellfish by Food Standards Scotland (FSS), and the concurrent monitoring of harmful phytoplankton in the water column acts as an important early warning system of future shellfish toxin contaminations. Since A. spinosum is very small (12-16 μm long) it is difficult to identify using a light microscope, therefore molecular techniques have been developed to detect species-specific environmental DNA from phytoplankton samples. In this thesis the application and verification of quantitative realtime polymerase chain reaction (qPCR) is discussed in detail and documents its first use in Scottish waters to survey A. spinosum abundance and seasonality. The limit of detection of the method was found to be 2000 ±5600 cells L-1, however it is unclear whether this is adequate for regulatory monitoring because it is not yet understood how cell density in the water column relates to AZA shellfish toxicity. The qPCR probe and primer sequences were also found to be too specific to detect all strains of the A. spinosum species, as new strains have been isolated since their development. This is a significant hindrance to the application of the tool for monitoring which will need to be addressed in the future through the isolation of local A. spinosum strains. Over a year long sampling period, A. spinosum was detected only twice (maximum cell density of 2545 ±5600 cells L-1, August 2014) off the Shetland Islands. The seasonality of the species in Scottish waters could not be assessed with so little data, however other observed harmful species of importance to shellfish regulatory monitoring are discussed; of particular note an unusual bloom of Dinophysis acuta as its association with a temperature front at the mouth of Loch Fyne. This thesis critiques the use of this qPCR technique for A. spinosum detection at high-throughput. The issues which have been highlighted do not prevent its future use by FSS, but highlight specific areas of development which need addressed before national monitoring can occur.
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