Characterisation of azaspiracid producers in New Zealand waters using novel molecular tools

Abstract

Azaspiracids (AZAs) are lipophilic polyether marine toxins produced by Azadinium and Amphidoma species. They can accumulate in several shellfish species and cause azaspiracid shellfish poisoning (AZP) in humans consuming contaminated seafood. The distribution of azaspiracids is worldwide but the culprit producer of these toxins could not always be identified. In New Zealand for instance, azaspiracids have been detected at low concentration in shellfish but the responsible organism remains unknown. However, the identification of this latter is a crucial requirement to significantly improve the monitoring efficiency of seafood safety programs as well as to ensure the protection of consumers. In this study, seawater samples collected as part of the New Zealand Marine Phytoplankton Monitoring Program have been screened for Azadinium and Amphidoma detection through real-time PCR (TaqMan and SYBR) and 2 unialgal cultures of Azadinium poporum have been established and entered the Cawthron Institute Culture Collection of Microalgae. The realtime PCR assays were highly sensitive and robust but the specificity of the TaqMan assays should be improved to provide more accurate results. The toxin production of the established cultures was analyzed by LC-MS/MS but currently regulated azaspiracids, i.e. AZA-1, -2, -3, were not detected. Sediment samples were also screened for Azadinium cysts and these latter were identified in samples collected in narrow embayments. This result highlighted the importance of including sediment analysis in monitoring programs for HABs early warning purposes. Cryopreservation trials on Azadinium cultures were also performed in order to bypass issues associated with serial subculturing and to ensure the long-term viability of the cultures. It has been argued that further trials should be carried out in ulterior studies to develop individually optimized protocols for each dinoflagellate species. In conclusion, this study drew the methodological outline for ulterior azaspiracid-related research topics, provided insight on the validation level of the used real-time PCR assays for Azadinium and Amphidoma detection and put forward several recommendations to improve our understanding on Azadinium distribution and associated toxin production. However, future studies are essential in order to bridge the gaps concerning the knowledge on this latter field of research, especially in New Zealand waters but also on a more global level.