Application of real-time PCR (QPCR) for enumeration of Planktothrix population (cyanobacteria) in the lake sediments : a paleolimnological approach

Fossilized organic components represent an archive of ancient aquatic microbial communities, and hence lake sediments can be used to reconstruct temporal variations in past community assemblages and biodiversity (Coolen et al., 2004). The recurrent presence of Planktothrix blooms has been observed in Lake Bourget since the 90s causing potential exposure of microcystins (MCs), the most prevalent cyanobacterial hepatotoxins in freshwaters. In this respect, the initial objectives of this research were to reconstruct the presence of Planktothrix spp. in lakesediments by conventional PCR, and then to evaluate their quantitative distribution along sediment core by real-time quantitative PCR (qPCR). Additionally, the changes in the assemblage of cyanobacteria, and more specifically, the possible genetic variants within Planktothrix sequences can be detected by sequencing the 16S and internal transcribed spacer (ITS) between the 16S and 23S rRNA regions. Recently, the TaqMan PCR or the Taq nuclease assay (TNA) was introduced to quantify specific genotypes of total or MC-producing cyanobacteria in the field (Kurmayer et al., 2004). In our research, TNA was used to quantify: (1) the total population of Planktothrix by either the intergenic spacer region within the phycocyanin (PC) operon (PC-IGS) (two primer/probe sets) or by 16S rDNA, and (2) MC-producing Planktothrix via mcyA region, which encodes one step in microcystin biosynthesis. The method was optimised, calibrated, specificity and reproducibility of the detection and possible inhibition by sediment components were also evaluated. Then, the multiple probes were combined to allow detection of more than one target in a single qPCR reaction. Due to the risk of contamination of sediment samples with modern DNA, special laboratory precautions were applied with all experiments. Results reveal that designed qPCR assay could be successfully applied to the analysis of the 30 first cm of Lake Bourget core. QPCR assays to quantify PC, 16S and mcyA genes of Planktothrix were considered specific and robust enough to prevent false positive results, and qPCR reactions were not inhibited by co-extracted impurities within the DNA extracts. Planktothrix DNA concentration in the sediments increases and gets the highest for depths corresponding to the lake transition from natural oligotrophic conditions to human induced eutrophication (1950s), but is low at depth corresponding to the depths of maximum eutrophication. The most recent samples, corresponding to the 2006-2008 time period, also show a very high concentration of Planktothrix. Results suggest that Planktothrix abundance in Lake Bourget is the highest for mesotrophic conditions. Further studies on the application of qPCR for sediment samples, as well as development of the dataset of sequencing on 16S-ITS region especially in depths where high Planktothrix counts were detected should be conducted.