qPCR assays for Alexandrium fundyenseand A. ostenfeldii(Dinophyceae) identified from Alaskan waters and a review of species-specific Alexandriummolecular assays

Abstract:Paralytic shellfish poisoning (PSP) poses a serious health threat in Alaska and prevents effective utilization of shellfish resources by subsistence and recreational harvesters. Substantial economic losses also affect shellfish growers during PSP events. The toxins responsible for PSP are produced by dinoflagellates in the genus Alexandrium. Despite the persistent threat posed by PSP and the long history of shellfish toxicity research, there is still confusion concerning the Alexandriumspecies that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandriumspecies present in Alaska and to develop polymerase chain reaction (PCR) assays for use in screening phytoplankton and sediment samples. Before developing the PCR assays for this study, we evaluated published assays and many were not adequate because of primer dimer formation or because of cross-reactivity. Rather than continue to grapple with the uncertainty and inadequacy of published assays, we developed new assays for the Alexandriumspecies most likely to be present in Alaska. Only Alexandrium fundyenseGroup I and A. ostenfeldiiwere identified from four sampling regions from southeast Alaska to Kodiak Island, indicating that these two species are widely distributed. PCR assays for these two species were converted to quantitative (q)PCR format for use in monitoring programs. During the course of this study, we realized that a systematic evaluation of all published (~150) Alexandriumspecies-specific assays would be of benefit. Toward this objective, we collated published AlexandriumPCR, qPCR, and in situhybridization assay primers and probes that targeted the small-subunit (SSU), internal transcribed spacer (ITS/5.8S), or D1–D3 large-subunit (LSU) (SSU/ITS/LSU) ribosomal DNA genes. Each individual primer or probe was screened against the GenBank database and Alexandriumgene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandriumassays that can be recommended for evaluation by the global harmful algal bloom community.