Detection of cyanobacterial sxt genes and paralytic shellfish toxins in freshwater lakes and brackish waters on Åland Islands, Finland

Savela, Henna; Spoof, Lisa; Perälä, Niina; Preede, Mikko; Lamminmäki, Urpo; Nybom, Sonja; Häggqvist, Kerstin; Meriluoto, Jussi; Vehniäinen, Markus

doi:10.1016/j.hal.2015.04.005

Cited by 35 publications

(31 citation statements)

References 55 publications

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“…Successful amplification resulted in 648 bp, 519 bp and 382 bp PCR products, respectively. The specificity N o n -c o m m e r c i a l u s e o n l y of sxtA and sxtG primers has been validated previously (Savela et al, 2015). For this study, the in silico specificity of sxtS primers was confirmed by running a query against the GenBank nr database (as available in February 2011) using blastn 2.2.29+ for highly similar sequences (Altschul et al, 1997) and optimized for short input sequences.…”

Section: Sample Preparation For Molecular Analysesmentioning

confidence: 88%

“…NH-5 (EU603710) sxtS gene and bases 216-597 of the C. raciborskii T3 (DQ787200) sxtS gene. The in vitro specificity was confirmed by analysing PST-producing and non-producing cyanobacterial strains following the same procedures as for sxtA and sxtG (Savela et al, 2015). For environmental sample analysis, sxtA, sxtG and sxtS-specific PCR reactions (total volume 20 µL) contained 1X Phire Reaction buffer and 0.4 µL Phire II HotStart DNA polymerase (Thermo Scientific), 0.2 mM dNTPs (Bioline), 0.5 µM appropriate forward and reverse primers and 4 µL sample lysate.…”

Section: Sample Preparation For Molecular Analysesmentioning

confidence: 99%

“…Several studies on European cyanobacteria have been carried out on strains isolated from freshwater lakes and reservoirs, and many have targeted the PST biosynthesis gene sxtA showing that while all PST-producing strains do carry the gene, it can also be found in Anabaena, Anabaenopsis and Aphanizomenon strains which appear to lack the ability to produce the toxins (Ballot et al, 2010;Ledreux et al, 2010;Cires et al, 2014). Other sxt genes, including sxtG, sxtB, sxtI and sxtX, have been studied to a lesser extent , Gkelis and Zaoutsos, 2014, Savela et al, 2015. Only a few studies have investigated the co-occurrence of sxt genes and PSTs in environmental samples in Europe (Gkelis and Zaoutsos, 2014;Savela et al, 2015) or elsewhere in the world (Al-Tebrineh et al, 2012;Bowling et al, 2013).…”

mentioning

confidence: 99%

“…Other sxt genes, including sxtG, sxtB, sxtI and sxtX, have been studied to a lesser extent , Gkelis and Zaoutsos, 2014, Savela et al, 2015. Only a few studies have investigated the co-occurrence of sxt genes and PSTs in environmental samples in Europe (Gkelis and Zaoutsos, 2014;Savela et al, 2015) or elsewhere in the world (Al-Tebrineh et al, 2012;Bowling et al, 2013).…”

mentioning

confidence: 99%

“…However, neither the prevalence of potentially PST-producing cyanobacteria, indicated by the presence of biosynthesis genes (sxt), nor PSTs in Polish lakes has been previously studied (Kobos et al, 2013). In this study, qualitative PCR methods targeting the sxtA and sxtG genes and a quantitative method for the detection of sxtB, which have been previously employed in the analysis of lakes and brackish coastal waters in Åland Islands, Finland (Savela et al, 2015) were applied to investigate freshwater lakes in western Poland. Targeting previously studied genes allowed comparisons to existing data.…”

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confidence: 99%

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

et al. 2017

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In central and southern Europe, Aphanizomenon spp., A. gracile Lemmermann in particular, have been associated with paralytic shellfish toxin (PST) production. In western Poland, A. gracile is very common, and Cylindrospermopsis raciborskii (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 sxtB copy numbers, sxtA, sxtG and sxtS 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 sxtA, sxtG and sxtS was determined using conventional qualitative PCR. Quantitative PCR (qPCR) was used to measure sxtB 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 sxtB gene were present in 23.5% of all samples (n=16) and in 14 lakes of the studied 34. Gene copy numbers ranged from 8.2×104 to 5.1×107 sxtB copies L-1 (mean 3.8×106). The median was 4.5×105 sxtB gene copies L-1 and the majority of results clustered at the lower end of the sxtB qPCR linear range. In 12 out of the 16 samples positive for sxtB the gene co-occurred with the other three targeted PST biosynthesis genes sxtA, sxtG and sxtS. 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-1, i.e., close to or below the quantitative detection limit of the HPLC-FLD method (0.01 µg L-1). One sample contained 0.57 µg L-1 saxitoxin, co-occurring with all four sxt genes studied. No correlation between PST and sxt gene occurrence or copy numbers was observed. A. gracile and C. raciborskii occurred in 92% and 50% of samples, respectively, containing PSTs, sxt 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 sxt genes targeted in this study could serve as a reliable marker for active PST biosynthesis.

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Section: Sample Preparation For Molecular Analysesmentioning

confidence: 88%

Section: Sample Preparation For Molecular Analysesmentioning

confidence: 99%