Application of real-time PCR to estimate toxin production by the cyanobacterium Planktothrix sp

Abstract

Quantitative real-time PCR methods are increasingly being applied for the enumeration of toxic cyanobacteria in the environment. However, to justify the use of real-time PCR quantification as a monitoring tool, significant correlations between genotype abundance and actual toxin concentrations are required. In the present study, we aimed to explain the concentrations of three structural variants of the hepatotoxin microcystin (MC) produced by the filamentous cyanobacterium Planktothrix sp., [Asp, butyric acid (Dhb)]-microcystin-RR (where RR means two arginines), [Asp, methyl-dehydro-alanine (Mdha)]-microcystin-RR, and [Asp, Dhb]-microcystin-homotyrosine-arginine (HtyR), by the abundance of the microcystin genotypes encoding their synthesis. Three genotypes of microcystin-producing cyanobacteria (denoted the Dhb, Mdha, and Hty genotypes) in 12 lakes of the Alps in Austria, Germany, and Switzerland from 2005 to 2007 were quantified by means of real-time PCR. Their absolute and relative abundances were related to the concentration of the microcystin structural variants in aliquots determined by high-performance liquid chromatography (HPLC). The total microcystin concentrations varied from 0 to 6.2 microg liter(-1) (mean +/- standard error [SE] of 0.6 +/- 0.1 microg liter(-1)) among the samples, in turn resulting in an average microcystin content in Planktothrix of 3.1 +/- 0.7 microg mm(-3) biovolume. Over a wide range of the population density (0.001 to 3.6 mm(3) liter(-1) Planktothrix biovolume), the Dhb genotype and [Asp, Dhb]-MC-RR were most abundant, while the Hty genotype and MC-HtyR were found to be in the lowest proportion only. In general, there was a significant linear relationship between the abundance/proportion of specific microcystin genotypes and the concentration/proportion of the respective microcystin structural variants on a logarithmic scale. We conclude that estimating the abundance of specific microcystin genotypes by quantitative real-time PCR is useful for predicting the concentration of microcystin variants in water.

FIG. 1.

Phytoplankton compositions in the study lakes classified into three groups depending on the average proportions of Planktothrix in the total phytoplankton biovolume (n = 5): <10% of the average proportion of Planktothrix in total phytoplankton, e.g., Schwarzensee (A); 10 to 50%, e.g., Mondsee (B), and 50 to 90%, e.g., Wörthersee (C). Phytoplankton groups that contributed ≤5% to the total biovolume are shown as “others.”

FIG. 2.

Relationship between the total Planktothrix biovolume (mm³ liter⁻¹) determined by real-time PCR via 16S rRNA genes and the microcystin genotypes consisting of the Mdha and Dhb genotypes (synthesizing either Mdha or Dhb in position 7 of the microcystin molecule). For details on the regression curves, see the text.

FIG. 3.

(A) Relationship between the total of the microcystin genotypes (Mdha and Dhb) determined by real-time PCR (biovolume in mm³ liter⁻¹) and the total microcystin concentration estimated by HPLC (μg liter⁻¹) from integrated (white symbols) and net (gray symbols) samples. Broken lines indicate 95% confidence limits. (B) Relationship between each of the microcystin genotypes and the corresponding microcystin variants for the same data set. For details on the regression curves, see the text.

FIG. 4.

(A) Proportions of genotypes encoding the synthesis of a specific microcystin variant (Mdha, Dhb, and Hty) revealed by real-time PCR (T) and of the respective microcystin structural variants revealed by HPLC (H). Box plots show the median and 25% to 75% percentiles. (B) Relationship between the average proportion (±SE) of microcystin variants determined by HPLC and the average proportion (±SE) of microcystin genotypes estimated by real-time PCR for each lake. For details on the regression curve, see the text.