Development and Application of a Quantitative PCR Assay to Assess Genotype Dynamics and Anatoxin Content in Microcoleus autumnalis-Dominated Mats

Microcoleus is a filamentous cyanobacteria genus with a global distribution. Some species form thick, cohesive mats over large areas of the benthos in rivers and lakes. In New Zealand Microcoleus autumnalis is an anatoxin producer and benthic proliferations are occurring in an increasing number of rivers nationwide. Anatoxin content in M. autumnalis-dominated mats varies spatially and temporally, making understanding and managing proliferations difficult. In this study a M. autumnalis-specific TaqMan probe quantitative PCR (qPCR) assay targeting the anaC gene was developed. The assay was assessed against 26 non-M. autumnalis species. The assay had a detection range over seven orders of magnitude, with a limit of detection of 5.14 x 10(-8) ng L-1. The anaC assay and a cyanobacterial specific 16S rRNA qPCR were then used to determine toxic genotype proportions in 122 environmental samples collected from 19 sites on 10 rivers in New Zealand. Anatoxin contents of the samples were determined using LC-MS/MS and anatoxin quota per toxic cell calculated. The percentage of toxic cells ranged from 0 to 30.3%, with significant (p < 0.05) differences among rivers. The anatoxin content in mats had a significant relationship with the percentage of toxic cells (R-2 = 0.38, p < 0.001), indicating that changes in anatoxin content in M. autumnalis-dominated mats are primarily related to the dominance of toxic strains. When applied to more extensive samples sets the assay will enable new insights into how biotic and abiotic parameters influence genotype composition, and if applied to RNA assist in understanding anatoxin production.