First report of cyanobacterial paralytic shellfish toxin biosynthesis genes and paralytic shellfish toxin production in Polish freshwater lakes

Abstract

<p>In central and southern Europe, <em>Aphanizomenon</em> spp., <em>A. gracile</em> Lemmermann in particular, have been associated with paralytic shellfish toxin (PST) production. In western Poland, <em>A. gracile</em> is very common, and <em>Cylindrospermopsis raciborskii</em> (Woloszyńska) Seenayya & Subba Raju, another potentially PST-producing species, is often found as well. To date it is, however, unknown if the cyanobacterial populations in this area harbour the genetic capability to produce PSTs, and to what extent toxin biosynthesis occurs. The objective of this study was to survey the prevalence of potentially PST-producing cyanobacteria by measuring paralytic shellfish toxin biosynthesis gene <em>sxtB</em> copy numbers, <em>sxtA</em>, <em>sxtG</em> and <em>sxtS</em> gene presence, and PST concentrations in Polish freshwater lakes. In total, 34 lakes in western Poland were sampled twice during summer 2010. The presence of PST biosynthesis genes <em>sxtA</em>, <em>sxtG</em> and <em>sxtS</em> was determined using conventional qualitative PCR. Quantitative PCR (qPCR) was used to measure <em>sxtB </em>copy numbers, and the samples were analysed for PSTs using ion-pair high performance liquid chromatography with post-column oxidation and fluorescence detection (HPLC-FLD). Cyanobacteria carrying the <em>sxtB</em> gene were present in 23.5% of all samples (n=16) and in 14 lakes of the studied 34. Gene copy numbers ranged from 8.2×10<sup>4</sup> to 5.1×10<sup>7</sup> <em>sxtB</em> copies L<sup>-1</sup> (mean 3.8×10<sup>6</sup>). The median was 4.5×10<sup>5</sup> <em>sxtB</em> gene copies L<sup>-1</sup> and the majority of results clustered at the lower end of the <em>sxtB</em> qPCR linear range. In 12 out of the 16 samples positive for <em>sxtB</em> the gene co-occurred with the other three targeted PST biosynthesis genes <em>sxtA</em>, <em>sxtG</em> and <em>sxtS</em>. However, five additional samples lacked one or two of the targeted four genes. Thirteen samples contained PSTs, of which 12 samples at levels <0.072 µg L<sup>-1</sup>, <em>i.e.</em>, close to or below the quantitative detection limit of the HPLC-FLD method (0.01 µg L<sup>-1</sup>). One sample contained 0.57 µg L<sup>-1</sup> saxitoxin, co-occurring with all four <em>sxt</em> genes studied. No correlation between PST and <em>sxt</em> gene occurrence or copy numbers was observed. <em>A. gracile</em> and <em>C. raciborskii</em> occurred in 92% and 50% of samples, respectively, containing PSTs, <em>sxt</em> genes or both. In conclusion, the results confirm that potential PST producers constitute an established subpopulation of cyanobacteria in Polish freshwater lakes. However, none of the <em>sxt</em> genes targeted in this study could serve as a reliable marker for active PST biosynthesis.</p>

Protected Freshwater Ecosystem with Incessant Cyanobacterial Blooming Awaiting a Resolution

“…Major deletions events in MC-coding gene cluster were previously observed in non-toxic Planktothrix strains [30], suggesting that similar events may occur in STX-producing strains. Similarly to this study, the lack of the some of the targeted STX genes was previously observed by Savela et al (2017) in Polish lakes. As STXs were not measured directly, conclusions on correlation between gene presence/absence and toxin production cannot be made.…”

“…The STX sxtA coding gene was not amplified in this study. Since the specificity of sxtA, sxtG and sxtS primers has been previously validated by Savela et al [19,20], the lack of targeted genes may be due to unexpected sequence dissimilarities between primers and target that can occur in natural populations. Large-scale gene mutations such as deletions and insertions resulting in non-functional gene clusters may also cause a lack of PCR amplification.…”

The Diversity of Cyanobacterial Toxins on Structural Characterization, Distribution and Identification: A Systematic Review

“…ATXs were found in Ireland [241], Italy [242], France [243], Netherlands [244], Denmark [245], Bulgaria [219], Finland [204], Poland [246], Sweden [215], Portugal [220], Spain [247], the UK [248] and Germany [249]. STXs were reported in Portugal [250], Denmark [251], France [252], Germany [253], Czech [213], Italy [254], Finland [255], the UK [256], Poland [257], Bulgaria [219], Serbia [223], Greece [235], Russia [27], Norway [258] and Spain [258]. NODs were found in Latvia [227], Germany [228], Spain [259] and France [260].…”

Cyanotoxins Occurrence in Portugal: A New Report on Their Recent Multiplication

“…The lack of amplification of sxtA could be due to the absence in the filtered biomass of this gene or also due to the lack of the saxitoxin cyanobacterium producer in the same sample. Previous environmental analysis in Polish lakes applying these three genetic markers showed that sxtA can be negative in environmental samples [25]. Authors from this study suggested that the lack of amplifications could be due to either sequence dissimilarities between targets and primers, although sxtA is based in Chrysosporum sp.…”

“…Authors from this study suggested that the lack of amplifications could be due to either sequence dissimilarities between targets and primers, although sxtA is based in Chrysosporum sp. DNA sequences, or due to mutations (insertions and deletions) in the sxt gene cluster which would result in a negative amplification [25]. Since Portuguese samples were all negative for sxtA, we suggest that these negative results could be mainly attributed to insertions and deletions in the DNA sequences of the sxt gene cluster of North and Center Chrysosporum sp.…”