Quantitative Real-Time PCR for Determination ofMicrocystin Synthetase E Copy Numbers forMicrocystisandAnabaenainLakes

Abstract

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.

Variation of Microcystis and microcystins coupling nitrogen and phosphorus nutrients in Lake Erhai, a drinking-water source in Southwest Plateau, China

“…The mcy gene cluster encodes multifunctional peptide synthetases for MC biosynthesis in MC-producing species (Tillett et al 2000). Thus, toxic and non-toxic Microcystis abundances could be quantified by quantitative real-time PCR (qPCR) detecting mcy genes (Kurmayer et al 2002;Vaitomaa et al 2003;Via-Ordorika et al 2004;Xu et al 2010;Lin et al 2011).…”

Benthic cyanobacteria of the genus Nodularia are non-toxic, without gas vacuoles, able to glide and genetically more diverse than planktonic Nodularia

“…Region rbcLX (about 900 bp) was amplified with primers CW and DW and the PCR cycling conditions of Rudi et al (1998). An approximately 800 bp region of ndaF of Nodularia was amplified with primers and PCR originally designed for mcyE (microcystin synthetase subunit E gene) from Anabaena (Vaitomaa et al, 2003) with the exception of annealing at 52 uC. A fragment of about 252-350 bp of gvpA-IGS was amplified with primers PVAIF2 and PVAR2 (Barker et al, 1999) Edwards et al (1989).…”

“…These results show that the presence of ndaF is a good indicator of nodularin production. In the future, ndaF from Nodularia, similarly to mcyA and mcyE from Anabaena and Microcystis strains (Vaitomaa et al, 2003;Tillet et al, 2001), may prove to be a valuable tool for detection and early warning tests of nodularin-producers in water environments. The sequence divergence of ndaF among N. spumigena and Nodularia sp.…”

Genetic analysis on Dolichospermum (Cyanobacteria; sensu Anabaena) populations based on the culture-independent clone libraries revealed the dominant genotypes existing in Lake Taihu, China

“…), but also synthesize and release odor compounds (2-methylisoborneol (2-MIB), Geosmin) (Briand et al, 2008;Graham et al, 2010;Wang et al, 2011). Among the water bloom forming cyanobacterial genera, Microcystis, Planktothrix, Dolichospermum (formerly as planktonic species in Anabaena genus) (Wacklin et al, 2009), and Aphanizomenon are usually the rulers governing the freshwaters and producing numerous bioactive secondary metabolites over the world (Pan et al, 2009;Vaitomaa et al, 2003).…”