qPCR assays for Alexandrium fundyense and A. ostenfeldii (Dinophyceae) identified from Alaskan waters and a review of species-specific Alexandrium molecular 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 Alexandrium species that cause PSP in Alaska. The primary objective of this study was to identify the toxic Alexandrium species 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 Alexandrium species most likely to be present in Alaska. Only Alexandrium fundyense Group I and A. ostenfeldii were 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) Alexandrium species-specific assays would be of benefit. Toward this objective, we collated published Alexandrium PCR, qPCR, and in situ hybridization 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 Alexandrium gene sequence alignments constructed as part of this study. These data were used to identify a suite of species-specific Alexandrium assays that can be recommended for evaluation by the global harmful algal bloom community.

Key Words::

• Alexandrium catenella
• Alexandrium minutum
• Alexandrium monilatum
• Alexandrium pacificum
• Alexandrium tamarense
• Alexandrium monitoring
• Gulf of Alaska
• Paralytic shellfish poisoning (PSP)
• qPCR assay
• Saxitoxin

ACKNOWLEDGEMENTS

We thank two anonymous reviewers who provided constructive comments. Reference to trade names do not imply product endorsement by the National Ocean Service (NOS), NOAA. Funding was provided by the National Center for Coastal Ocean Science (NCCOS), NOAA and North Pacific Research Board Project 1118. This is contribution #5231 from University of Maryland Center for Environmental Science (UMCES), #16-179 for University of Maryland Institute of Marine and Environmental Technology (IMET), and #ECO867 from the Ecology and Oceanography of Harmful Algal Blooms (ECOHAB) program. This research was also funded in part by grants from Oceans and Human Health National Institutes of Health R01ES021949-01/NSFOCE1313888 and NOAA-NOS- NCCOS-2012-2002987 to ARP.

SUPPLEMENTARY DATA

Supplementary data associated with this article can be found online at http://dx.doi.org/10.2216/16-41.1.s1.