The Production of Yessotoxin by Protoceratium Reticulatum

Abstract

Yessotoxin, a marine biotoxin, has been responsible for several shellfish toxicity events in New Zealand. The dinoflagellate Protoceratium reticulatum was thought to be responsible for the occurrence of yessotoxin in these events. In phytoplankton monitoring it is crucial to know at what stage of a bloom toxicity will be the greatest. It is also very important to know what derivatives of yessotoxin are produced in certain conditions and the contribution these derivatives make to the total toxicity enabling biotoxin assays to be designed around what specifically needs to be detected. The overall aim of this thesis was to establish what conditions will favour the formation of P. reticulatum blooms and what conditions promote yessotoxin or yessotoxin analogue production by P. reticulatum. This was investigated using a local unialgal isolate of P. reticulatum and growing it in batch culture under controlled environmental conditions. The optimal conditions for growth of P. reticulatum and toxin production were investigated. Of the environmental conditions tested, those that best promoted the growth of P. reticulatum were a temperature of 21 °C, irradiance levels of between 25 and 50 μmol m-2 s-1, salinity level of the seawater of 35 ‰ and daylength in a cycle of 12:12 h light:dark. The algal growth medium that best promoted growth was GP medium. YTX production per cell was monitored throughout the growth cycle and levels were observed to typically increase as the cell cycle progressed. YTX production per cell was noted to vary depending on the environmental conditions that the cultures were maintained in. The conditions tested that gave the greatest toxin production were not necessarily the same as the best conditions for growth. Of the environmental conditions tested, those that promoted the greatest toxin production by P. reticulatum were a temperature of 21 °C, an irradiance level of 50 μmol m-2 s-1, salinity level of the culture medium of 15 ‰ and daylength in a cycle of 14:10 h light:dark. When the nutrient regime was altered toxin production did not alter markedly. Analogues of YTX were not detected in any cultures of P. reticulatum under the range of conditions tested, except for the culture containing limited nitrate and phosphate. The analogue was detected using HPLC and its identity was not elucidated. Preliminary HPLC evidence was found to support the hypothesis that bacteria associated with P. reticulatum could produce YTX or YTX analogues but this was not confirmed. Although this thesis does not include an investigation of the natural environment, in which conditions fluctuate and other impacting species are present, the research has identified the physical and chemical conditions likely to favour a bloom of this dinoflagellate. In showing that toxin production continues throughout the growth cycle, the research also indicates that total toxicity increases as the growth cycle progresses. This thesis therefore serves as a good basis for predicting when blooms of P. reticulatum are most likely to occur, and when they are likely to be the most toxic.