sxtA-based quantitative molecular assay to identify saxitoxin-producing harmful algal blooms in marine waters

Abstract

The recent identification of genes involved in the production of the potent neurotoxin and keystone metabolite saxitoxin (STX) in marine eukaryotic phytoplankton has allowed us for the first time to develop molecular genetic methods to investigate the chemical ecology of harmful algal blooms in situ. We present a novel method for detecting and quantifying the potential for STX production in marine environmental samples. Our assay detects a domain of the gene sxtA that encodes a unique enzyme putatively involved in the sxt pathway in marine dinoflagellates, sxtA4. A product of the correct size was recovered from nine strains of four species of STX-producing Alexandrium and Gymnodinium catenatum and was not detected in the non-STX-producing Alexandrium species, other dinoflagellate cultures, or an environmental sample that did not contain known STX-producing species. However, sxtA4 was also detected in the non-STX-producing strain of Alexandrium tamarense, Tasmanian ribotype. We investigated the copy number of sxtA4 in three strains of Alexandrium catenella and found it to be relatively constant among strains. Using our novel method, we detected and quantified sxtA4 in three environmental blooms of Alexandrium catenella that led to STX uptake in oysters. We conclude that this method shows promise as an accurate, fast, and cost-effective means of quantifying the potential for STX production in marine samples and will be useful for biological oceanographic research and harmful algal bloom monitoring.

Fig. 1.

Phytoplankton and S. glomerata sampling sites. (A) New South Wales (in white), Australia, and the sites in three estuaries in which A. catenella blooms were sampled; (B) Brisbane Water; (C) the Georges River; (D) Wagonga Inlet. The scale bar in panel D represents 2 km in inset maps B, C, and D.

Fig. 2.

Standard curve of the sxtA4 primer pair based on replicate dilutions (± SD) of DNA from known numbers of cells of three exponentially growing Alexandrium catenella strains, ACSH02, ACTRA01, and ACCC01. The assay was tested using DNA concentrations representing 30 to 2,600 cells. The regression equations are shown.

Fig. 3.

Abundance of sxtA4 gene copies (primary y axis) and estimates of Alexandrium catenella cells (secondary y axis) based on cell identifications and counts using a microscope, as well as cell enumeration using LSU rDNA qPCR, at the Georges River (a) or Wagonga Inlet (b) sampling site during November 2010.