Paralytic shellfish poisoning toxin-producing cyanobacterium Aphanizomenon gracile in northeast Germany

Abstract

Neurotoxic paralytic shellfish poisoning (PSP) toxins, anatoxin-a (ATX), and hepatotoxic cylindrospermopsin (CYN) have been detected in several lakes in northeast Germany during the last 2 decades. They are produced worldwide by members of the nostocalean genera Anabaena, Cylindrospermopsis, and Aphanizomenon. Although no additional sources of PSP toxins and ATX have been identified in German water bodies to date, the observed CYN concentrations cannot be produced solely by Aphanizomenon flos-aquae, the only known CYN producer in Germany. Therefore, we attempted to identify PSP toxin, ATX, and CYN producers by isolating and characterizing 92 Anabaena, Aphanizomenon, and Anabaenopsis strains from five lakes in northeast Germany. In a polyphasic approach, all strains were morphologically and phylogenetically classified and then tested for PSP toxins, ATX, and CYN by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) and screened for the presence of PSP toxin- and CYN-encoding gene fragments. As demonstrated by ELISA and LC-MS, 14 Aphanizomenon gracile strains from Lakes Melang and Scharmützel produced four PSP toxin variants (gonyautoxin 5 [GTX5], decarbamoylsaxitoxin [dcSTX], saxitoxin [STX], and neosaxitoxin [NEO]). GTX5 was the most prevalent PSP toxin variant among the seven strains from Lake Scharmützel, and NEO was the most prevalent among the seven strains from Lake Melang. The sxtA gene, which is part of the saxitoxin gene cluster, was found in the 14 PSP toxin-producing A. gracile strains and in 11 non-PSP toxin-producing Aphanizomenon issatschenkoi, A. flos-aquae, Anabaena planktonica, and Anabaenopsis elenkinii strains. ATX and CYN were not detected in any of the isolated strains. This study is the first confirming the role of A. gracile as a PSP toxin producer in German water bodies.

FIG. 1.

Ratio (%) of PSP toxin variants in 14 A. gracile strains from Lakes Scharmützel and Melang, as determined with LC-MS/MS.

FIG. 2.

Maximum likelihood tree based on partial sxtA sequences of 30 cyanobacterial strains. Strains from this study are marked in bold. Bootstrap values above 50 are included. The bar indicates 1% sequence divergence. +, PSP toxin producer; −, PSP toxin not detected; *, accession number listed in Table 2.

FIG. 3.

Micrographs of Nostocales strains investigated in this study. (a) Aphanizomenon flos-aquae; (b) Aphanizomenon gracile; (c) Aphanizomenon issatschenkoi; (d) Anabaena bergii; (e) Anabaena crassa; (f) Anabaena flos-aquae; (g) Anabaena lemmermanii; (h) Anabaena planktonica; and (i) Anabaenopsis elenkinii. Scale bars indicate 25 μm.

FIG. 4.

Maximum likelihood tree based on partial PC-IGS sequences of 107 cyanobacterial strains. Strains from this study are marked in bold. Bootstrap values above 50 are included. The scale bar indicates 10% sequence divergence. L, Lake Langer; M, Lake Melang; S, Lake Scharmützel. +, PSP toxin producer; −, PSP toxin not detected; *, accession number listed in Table 2 (and see Table S1 in the supplemental material).