Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1

Shauna A Murray,Gurjeet S Kohli,Donald M Anderson,Michael L Brosnahan,Rendy Ruvindy

doi:10.1038/s41598-019-51074-3

Shauna A Murray, Gurjeet S Kohli + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-51074-3

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Abstract

Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L−1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.

Highlights

Saxitoxin (STX) and its many analogues, known as the paralytic shellfish toxins (PSTs), are neurotoxins that can have profound effects on marine food chains[1] and are responsible for causing the syndrome paralytic shellfish poisoning (PSP) in humans
All potential PST-producing species of Alexandrium, Pyrodinium bahamense and Gymnodinium catenatum can be counted in a single assay, without detecting non-STX producing species which may be important in regions where closely related toxic and non-toxic Alexandrium species co-occur
The Quantitative polymerase chain reaction (qPCR) assay based on sxtA4 was found to be 93–94%

Summary

Introduction

Saxitoxin (STX) and its many analogues, known as the paralytic shellfish toxins (PSTs), are neurotoxins that can have profound effects on marine food chains[1] and are responsible for causing the syndrome paralytic shellfish poisoning (PSP) in humans. STX-producing phytoplankton comprise ~8 species of the genus Alexandrium, as well as Gymnodinium catenatum and Pyrodinium bahamense[2,3,4,5,6]. Because these blooms threaten seafood safety, they are monitored extensively through a variety of frequently labour-intensive methods (reviewed in[7]). Www.nature.com/scientificreports domains, and shows two isoforms: one of which comprises four domains, sxtA1–sxtA4, while the other one encompasses only the domains sxtA1–sxtA329, and may represent a duplicated, paralogous copy of sxtA1 The latter sxtA1-A3 isoform is present in several species of dinoflagellates that do not produce PSTs30. It has been shown that, at least within the species Alexandrium minutum, the copy number of sxtA4 may be correlated to the amount of PSTs produced different strains[39]

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