Pilot search for cylindrospermopsin-producers in nine shallow Bulgarian waterbodies reveals nontoxic strains of Raphidiopsis raciborskii, R. mediterranea and Chrysosporum bergii

This paper presents results from the first application of molecular-genetic analysis combined with conventional light microscopy (LM) for identification of cylindrospermopsin (CYN) producers in field phytoplankton samples from Bulgaria. In total 68 cyanoprokaryotes were found by LM, out of which 18 were considered potential CYN-producers according to the literature. They occurred in different abundance (0-40%) in five waterbodies. The presence of CYN-producers was sought by application of the primer pair cynsulfF/cylnamR specific for the sulfotransferase gene (cyrJ) considered the best genetic marker for detection of CYN-toxigenic strains. Although CYN was detected by enzyme-linked immunosorbent assay (ELISA) in two waterbodies (lake Vaya and reservoir Mandra), we did not find cyanoprokaryotes with cyrJ gene. Therefore, Raphidiopsis raciborskii, despite being identified by LM in both waterbodies where CYN occurred, was not the producer of this toxin and belonged to the European nontoxic population of the species. The same was true for the less widely spread Raphidiopsis mediterranea and Chrysosporum bergii (both found in small amounts only in lake Vaya). Our results on CYN occurrence without the assignment of its producer in the collected samples suggest that: 1) CYN remains stable in water after disappearance of the toxigenic species, or, more probably, 2) besides the already known species, there are others that are associated with the production of CYN. Because revealing of novel CYN producers is of ultimate importance for health risk assessment and water management, we propose more analyses to be performed in future studies.