qPCR assays for the detection and quantification of multiple paralytic shellfish toxin-producing species of Alexandrium

<p>Paralytic shellfish toxin producing dinoflagellates have negatively impacted the shellfish aquaculture industry worldwide, including in Australia and New Zealand. Morphologically identical cryptic species of dinoflagellates that may differ in toxicity, in particular, species of the former <i>Alexandrium tamarense</i> species complex, co-occur in Australia, as they do in multiple regions in Asia and Europe. To understand the dynamics and the ecological drivers of the growth of each species in the field, accurate quantification at the species level is crucial. We have developed the first quantitative polymerase chain reaction (qPCR) primers for <i>A. australiense</i>, and new primers targeting <i>A. ostenfeldii, A. catenella</i>, and <i>A. pacificum</i>. We showed that our new primers for <i>A. pacificum</i> are more specific than previously published primer pairs. These assays can be used to quantify planktonic cells and cysts in the water column and in sediment samples with limits of detection of 2 cells/L for the <i>A. catenella</i> and <i>A. australiense</i> assays, 2 cells/L and 1 cyst/mg sediment for the <i>A. pacificum</i> assay, and 1 cells/L for the <i>A. ostenfeldii</i> assay, and efficiencies of >90%. We utilized these assays to discriminate and quantify co-occurring <i>A. catenella, A. pacificum</i>, and <i>A. australiense</i> in samples from the east coast of Tasmania, Australia.</p>