CONFIRMATION OF TOXIN-PRODUCING ABILITY IN A STRAIN OF MICROCYSTIS WESENBERGII

<italic>Microcystis</italic> blooms, increasingly frequent in lakes, reservoirs, and other aquatic ecosystems, raise concerns due to microcystins (MCs) production. <italic>Microcystis wesenbergii</italic>, one of the predominant bloom-forming species, has traditionally been considered a non-toxic strain. In this study, immunofluorescence analysis revealed positive reactivity of a purified <italic>M. wesenbergii</italic> strain (Hui-8) to the microcystin synthetase protein McyF nanobody, while molecular characterization confirmed its taxonomic identity as <italic>M. wesenbergii</italic> and revealed toxin-producing genes (<italic>mcyB</italic> and <italic>mcyD</italic>). Quantitative ELISA analysis indicated that the toxin production capacity of Hui-8 was lower than that of the toxic reference strain <italic>M. aeruginosa</italic> PCC 7806. Subsequent HPLC-MS analysis identified the toxin variant as MC-RR. MCs exposure impair chironomid larvae mobility, gut microbiota, and lipid metabolism; disrupt calcium and potassium homeostasis in submerged plants, causing osmotic stress and leaf damage; and induce rapid cell deformation, inactivation, or necrosis via PP1/PP2A inhibition, leading to cytoskeleton disintegration and organelle dysfunction with prolonged exposure. Most <italic>Microcystis</italic> species produce MCs, with <italic>M. aeruginosa</italic> extensively studied for high MC production. For <italic>M. wesenbergii</italic>, limited evidence—single toxin gene detection and animal experiments—leaves its toxicity inconclusive, and no toxin genes are reported in its genomic data, reinforcing its non-toxic label. However, using McyF nanobody targeting toxin synthesis gene, some <italic>M. wesenbergii</italic> strains showed positive results. This study analyzed laboratory-purified <italic>M. wesenbergii</italic> strains and identified one strain capable of producing microcystins. The colonial <italic>M. wesenbergii</italic> strains (Hui-8) used in this study were isolated, purified, and maintained under laboratory conditions with stable morphology for three years. Based on the typical <italic>M. wesenbergii</italic> strain (Hui-8), we compared its physiological differences with non-toxigenic <italic>M. wesenbergii</italic> and toxigenic <italic>M. aeruginosa.</italic> Through immunofluorescence localization, PCR detection of toxin genes (<italic>mcy</italic>B/<italic>mcy</italic>D), and ELISA quantification confirmed that <italic>M. wesenbergii</italic> (Hui-8) synthesizes microcystin-RR (MC-RR), challenging the traditional classification of this species as non-toxigenic. Using the McyF nanobody F4-EGFP, Hui-8 bound significantly to the fluorescent nanobody—similar to toxic <italic>M. aeruginosa</italic> PCC7806—unlike colonial <italic>M. wesenbergii</italic> TH29 and unicellular FACHB-929 suggesting potential toxicity. PCR confirmed <italic>mcy</italic>B and <italic>mcy</italic>D in Hui-8, with sequences matching GenBank records. Notably, while the McyF nanobody test was positive, PCR failed to amplify <italic>mcy</italic>F, <italic>mcy</italic>A, and <italic>mcy</italic>C, possibly due to sequence divergence from <italic>M. aeruginosa</italic>-derived primers or atypical gene organization in <italic>M. wesenbergii</italic>. PCR detection of <italic>mcy</italic>B and <italic>mcy</italic>D does not necessarily confirm toxin production. Therefore, an MCs ELISA kit was used. The results showed that Hui-8 has a certain toxin-producing capability, with a toxin concentration of approximately 1.44×10^–2 ng/mg, which is significantly lower than that of <italic>M. aeruginosa</italic> PCC7806 (6.8×10^–2 ng/mg). HPLC-MS identified a peak at 2.3min matching MC-RR standards, with another peak at approximately 5.2min suggesting a possible uncharacterized toxin variant.