Although cyanobacterial harmful algal blooms (CHABs) are promoted by nutrient loading and elevated temperatures, the effects of these processes on bloom diversity are unclear. This study used traditional and next-generation sequencing approaches to assess shifts in phytoplankton, cyanobacterial (16S rRNA), and microcystin-producing (mcyE) communities during CHABs in western Lake Erie (Maumee and Sandusky Bays) in response to natural and experimental gradients of nitrogen (N), phosphorus (P), and temperature. CHABs were most intense near the Maumee and Sandusky Rivers and were dominated by Microcystis and Planktothrix, respectively. Sequencing of 16S amplicons revealed cryptic cyanobacterial diversity (47 genera) including high abundances of two distinct clades of Synechococcus in both bays and significant differences in community structure between nutrient-rich nearshore sites and less eutrophic offshore sites. Sequencing of mcyE genes revealed low taxonomic (n = 3) but high genetic diversity (n = 807), with toxigenic strains of Planktothrix being more abundant than Microcystis and more closely paralleling microcystin concentrations. Cyanobacterial abundance significantly increased in response to elevated N, with the greatest increases in combined high N, P, and temperature treatments that concurrently suppressed green and brown algae. N significantly increased microcystin concentrations and the relative abundance of nondiazotrophic genera such as Planktothrix, while diazotrophic genera such as Dolichospermum and Aphanizomenon were, in some cases, enhanced by high P and temperature. While nutrients and elevated temperatures promote CHABs, differing combinations selectively promote individual cyanobacterial genera and strains, indicating management of both N and P will be required to control all cyanobacteria in Lake Erie, particularly as lake temperatures rise.

Microcystis and Anabaena (Dolichospermum) are among the most toxic cyanobacterial genera and often succeed each other during harmful algal blooms. The role allelopathy plays in the succession of these genera is not fully understood. The allelopathic interactions of six strains of Microcystis and Anabaena under different nutrient conditions in co-culture and in culture-filtrate experiments were investigated. Microcystis strains significantly reduced the growth of Anabaena strains in mixed cultures with direct cell-to-cell contact and high nutrient levels. Cell-free filtrate from Microcystis cultures proved equally potent in suppressing the growth of nutrient replete Anabaena cultures while also significantly reducing anatoxin-a production. Allelopathic interactions between Microcystis and Anabaena were, however, partly dependent on ambient nutrient levels. Anabaena dominated under low N conditions and Microcystis dominated under nutrient replete and low P during which allelochemicals caused the complete suppression of nitrogen fixation by Anabaena and stimulated glutathione S-transferase activity. The microcystin content of Microcystis was lowered with decreasing N and the presence of Anabaena decreased it further under low P and high nutrient conditions. Collectively, these results indicate that strong allelopathic interactions between Microcystis and Anabaena are closely intertwined with the availability of nutrients and that allelopathy may contribute to the succession, nitrogen availability, and toxicity of cyanobacterial blooms.

The toxic cyanobacterium Microcystis is one of the most pervasive harmful algal bloom (HAB) genera and naturally occurs in large colonies known to harbor diverse heterotrophic bacterial assemblages. While colony-associated microbiomes may influence Microcystis blooms, there remains a limited understanding of the structure and functional potential of these communities and how they may be shaped by changing environmental conditions. To address this gap, we compared the dynamics of Microcystis-attached (MCA), free-living (FL), and whole water (W) microbiomes during Microcystis blooms using next-generation amplicon sequencing (16S rRNA), a predictive metagenome software, and other bioinformatic approaches. Microbiomes were monitored through high resolution spatial-temporal surveys across two North American lakes, Lake Erie (LE) and Lake Agawam (LA; Long Island, NY, United States) in 2017, providing the largest dataset of these fractions to date. Sequencing of 126 samples generated 7,922,628 sequences that clustered into 7,447 amplicon sequence variants (ASVs) with 100% sequence identity. Across lakes, the MCA microbiomes were significantly different than the FL and W fractions being significantly enriched in Gemmatimonadetes, Burkholderiaceae, Rhizobiales, and Cytophagales and depleted of Actinobacteria. Further, although MCA communities harbored > 900 unique ASVs, they were significantly less diverse than the other fractions with diversity inversely related to bloom intensity, suggesting increased selection pressure on microbial communities as blooms intensified. Despite taxonomic differences between lakes, predicted metagenomes revealed conserved functional potential among MCA microbiomes. MCA communities were significantly enriched in pathways involved in N and P cycling and microcystin-degradation. Taxa potentially capable of N 2-fixation were significantly enriched (p < 0.05) and up to four-fold more abundant within the MCA faction relative to other fractions, potentially aiding in the proliferation of Microcystis blooms during low N conditions. The MCA predicted metagenomes were conserved over 8 months of seasonal changes in temperature and N availability despite strong

The bloom-forming, toxic cyanobacterium Microcystis synthesizes multiple secondary metabolites and has been shown to deter zooplankton grazing. However, the biochemical and/or molecular basis by which Microcystis deters zooplankton remains unclear. This global transcriptomic study explored the response of Microcystis to direct and indirect exposures to multiple densities of two cladoceran grazers, Daphnia pulex and D. magna. Higher densities of both daphnids significantly reduced Microcystis cell densities and elicited a stronger transcriptional response in Microcystis. While many putative grazer deterrence genes (encoding microcystin, aeruginosin, cyanopeptolin, and microviridin) were largely unaffected by zooplankton, transcripts for heat shock proteins (hsp) increased in abundance. Beyond metabolites and hsp, large increases in the abundances of transcripts from photosynthetic processes were observed, evidencing energy acquisition pathways were stimulated by grazing. In addition, transcripts of genes associated with the production of extracellular polysaccharides and gas vesicles significantly increased in abundance. These genes have been associated with colony formation and may have been invoked to deter grazers. Collectively, this study demonstrates that daphnid grazers induce a significant transcriptomic response in Microcystis, suggesting this cyanobacterium upregulates specific biochemical pathways to adapt to predation.IMPORTANCE This work explores the transcriptomic responses of Microcystis aeruginosa following exposure to grazing by two cladocerans, Daphnia magna and D. pulex. Contrary to previous hypotheses, Microcystis did not employ putative grazing deterrent secondary metabolites in response to the cladocerans, suggesting they may have other roles within the cell, such as oxidative stress protection. The transcriptional metabolic signature during intense grazing was largely reflective of a growth and stress response, although increasing abundances of transcripts encoding extracellular polysaccharides and gas vesicles were potentially related to predator avoidance.

Globally, eutrophication and warming of aquatic ecosystems has increased the frequency and intensity of cyanobacterial blooms and their associated toxins, with the simultaneous detection of multiple cyanotoxins often occurring. Despite the co-occurrence of cyanotoxins such as microcystins and anatoxin-a (ATX) in water bodies, their effects on phytoplankton communities are poorly understood. The individual and combined effects of microcystin-LR (MC-LR) and ATX on the cyanobacteria Microcystis spp., and Anabaena variabilis (a.k.a. Trichormus variabilis), and the chlorophyte, Selenastrum capricornutum were investigated in the present study. Cell density, chlorophyll-a content, and the maximum quantum efficiency of photosystem II (Fv/Fm) of Microcystis cells were generally lowered after exposure to ATX or MC-LR, while the combined treatment with MC-LR and ATX synergistically reduced the chlorophyll-a concentration of Microcystis strain LE-3. Intracellular levels of microcystin in Microcystis LE-3 significantly increased following exposure to MC-LR + ATX. The maximum quantum efficiency of photosystem II of Anabaena strain UTEX B377 declined during exposure to the cyanotoxins. Nitrogen fixation by Anabaena UTEX B377 was significantly inhibited by exposure to ATX, but was unaffected by MC-LR. In contrast, the combination of both cyanotoxins (MC-LR + ATX) caused a synergistic increase in the growth of S. capricornutum. While the toxins caused an increase in the activity of enzymes that scavenge reactive oxygen species in cyanobacteria, enzyme activity was unchanged or decreased in S. capricornutum. Collectively this study demonstrates that MC-LR and ATX can selectively promote and inhibit the growth and performance of green algae and cyanobacteria, respectively, and that the combined effect of these cyanotoxins was often more intense than their individual effects on some strains. This suggests that the release of multiple cyanotoxins in aquatic ecosystems, following the collapse of blooms, may influence the succession of plankton communities.