Nitrogen Limitation of Intense and Toxic Cyanobacteria Blooms in Lakes within Two of the Most Visited Parks in the USA: The Lake in Central Park and Prospect Park Lake.

The Lake in Central Park (LCP) and Prospect Park Lake (PPL) in New York City (NYC), USA, are lakes within two of the most visited parks in the USA. Five years of nearshore sampling of these systems revealed extremely elevated levels of cyanobacteria and the toxin, microcystin, with microcystin levels averaging 920 µg L−1 and chlorophyll a from cyanobacterial (cyano-chla) populations averaging 1.0 × 105 µg cyano-chla L−1. Both lakes displayed elevated levels of orthophosphate (DIP) relative to dissolved inorganic nitrogen (DIN) during summer months when DIN:DIP ratios were < 1. Nutrient addition and dilution experiments revealed that N consistently limited cyanobacterial populations but that green algae were rarely nutrient limited. Experimental additions of public drinking water that is rich in P and, to a lesser extent N, to lake water significantly enhanced cyanobacterial growth rates in experiments during which N additions also yielded growth enhancement. Collectively, this study demonstrates that the extreme microcystin levels during blooms in these highly trafficked lakes represent a potential human and animal health threat and that supplementation of these artificial lakes with public drinking water to maintain water levels during summer may promote the intensity and N limitation of blooms.

Nitrogen and phosphorus significantly alter growth, nitrogen fixation, anatoxin-a content, and the transcriptome of the bloom-forming cyanobacterium, Dolichospermum.

While freshwater cyanobacteria are traditionally thought to be limited by the availability of phosphorus (P), fixed nitrogen (N) supply can promote the growth and/or toxin production of some genera. This study characterizes how growth on N2 (control), nitrate (NO3 -), ammonium (NH4 +), and urea as well as P limitation altered the growth, toxin production, N2 fixation, and gene expression of an anatoxin-a (ATX-A) - producing strain of Dolichospermum sp. 54. The transcriptomes of fixed N and P-limited cultures differed significantly from those of fixed N-deplete, P-replete (control) cultures, while the transcriptomes of P-replete cultures amended with either NH4 + or NO3 - were not significantly different relative to those of the control. Growth rates of Dolichospermum (sp. 54) were significantly higher when grown on fixed N relative to without fixed N; growth on NH4 + was also significantly greater than growth on NO3 -. NH4 + and urea significantly lowered N2 fixation and nifD gene transcript abundance relative to the control while cultures amended with NO3 - exhibited N2 fixation and nifD gene transcript abundance that was not different from the control. Cultures grown on NH4 + exhibited the lowest ATX-A content per cell and lower transcript abundance of genes associated ATX-A synthesis (ana), while the abundance of transcripts of several ana genes were highest under fixed N and P - limited conditions. The significant negative correlation between growth rate and cellular anatoxin quota as well as the significantly higher number of transcripts of ana genes in cultures deprived of fixed N and P relative to P-replete cultures amended with NH4 + suggests ATX-A was being actively synthesized under P limitation. Collectively, these findings indicate that management strategies that do not regulate fixed N loading will leave eutrophic water bodies vulnerable to more intense and toxic (due to increased biomass) blooms of Dolichospermum.

Dynamic Responses of Endosymbiotic Microbial Communities Within Microcystis Colonies in North American Lakes to Altered Nitrogen, Phosphorus, and Temperature Levels.

The toxic cyanobacterium, Microcystis, is a pervasive cyanobacterial harmful algal bloom (CHAB) - forming genus that naturally occurs in colonies that harbor diverse microbiomes of heterotrophic bacteria. While the effects of nutrient loading and climatic warming on CHABs are well-known, little is known regarding how these environmental drivers alter the structural and functional potential of the microbial assemblages associated with blooms that, in turn, may impact cyanobacterial growth. Here, we used next-generation sequencing of 16S ribosomal rRNA genes to characterize the dynamics of the bacterial assemblages within Microcystis colonies in two temperate North American lakes: Lake Erie and Lake Agawam (NY, United States) and quantified their responses to experimentally increased levels of nitrogen (N), phosphorus (P) and temperature. Across experiments, Microcystis populations were consistently and significantly promoted by N and, to a lesser extent, elevated temperature (p < 0.05). In contrast, bacterial assemblages within Microcystis colonies were more resilient to environmental perturbations, with the relative abundance of 7-16% of amplicon sequence variants changing and several individual taxa displaying significant (p < 0.05) increases and decreases in relative abundance, primarily in response to elevated temperature and to a lesser extent, N. In contrast to individual taxa, community diversity was not significantly altered by individual treatments during experiments but rather was inversely correlated with the intensity of Microcystis blooms (p < 0.001). While predicted metabolic function was even less impacted by environmental drivers than microbial diversity, the predicted abundance of nitrogenase (nifH), alkaline phosphatase (phoX), and urease (ure) genes significantly increased in response to N but decreased in response to increased temperature (p < 0.05). Collectively, the resilience of microbial community structure and function within colonies suggests they may support the ability of Microcystis to persist through short-term fluctuations in environmental conditions by supplying essential nutrients.

The Composition and Function of Microbiomes Within Microcystis Colonies Are Significantly Different Than Native Bacterial Assemblages in Two North American Lakes.

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 N2-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 temporal succession in microbiome composition. Collectively, these findings indicate that Microcystis colonies harbor a statistically distinct microbiome with a conserved functional potential that may help facilitate bloom persistence under environmentally unfavorable conditions.

The Individual and Combined Effects of the Cyanotoxins, Anatoxin-a and Microcystin-LR, on the Growth, Toxin Production, and Nitrogen Fixation of Prokaryotic and Eukaryotic Algae.

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.

Succession and toxicity of Microcystis and Anabaena (Dolichospermum) blooms are controlled by nutrient-dependent allelopathic interactions.

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.

Effects of Microcystis on development of early life stage Japanese medaka (Oryzias latipes): Comparative toxicity of natural blooms, cultured Microcystis and microcystin-LR.

Freshwater cyanobacterial harmful algal blooms (CyanoHABs) caused by algae in the genus Microcystis have been increasing in frequency and severity in recent decades. Microcystis blooms threaten aquatic organisms through effects associated with the rapid increase of biomass and the production of the hepatotoxin microcystin (MC) by toxic strains. Among fish, effects of blooms are likely to be more severe for early life stages, and physiological impacts on this life stage could significantly impact recruitment and fish populations. This study explores the effects of Microcystis blooms on the development of fish using the model organism, the Japanese medaka (Oryzias latipes), under realistic exposure conditions. Medaka embryos were exposed to natural blooms collected from New York City (USA) lakes, lab cultures of Microcystis, and MC-LR solutions. Field collected samples were more toxic than lab cultures (even when compared at the same algal density or MC concentration), causing decreased survival, premature time to hatch, reduced body length, yolk sac edema, and decreased heart rate, while lab culture exposures only resulted in bradycardia. Heart rate was the most sensitive endpoint measured, being depressed in embryos exposed to both lab cultures and field collected blooms. Generalized linear model analysis indicated bradycardia was statistically associated with both cell densities of blooms and MC concentrations, while single factor analysis indicated that MC concentrations had a stronger correlation compared to cell densities. However, MC exposure could not fully explain the effects observed, as exposures to MC-LR solutions alone were not able to reduce heart rate as severely as algal exposures. Collectively, these experiments indicate that factors beyond exposure to MC or even isolated Microcystis strains influence heart rate of fish exposed to Microcystis blooms. Enhanced mortality, depressed heart rate, and abnormal development observed in response to environmentally realistic exposures of Microcystis blooms could affect success of fish at both individual or population levels.

Transcriptomic Responses in the Bloom-Forming Cyanobacterium Microcystis Induced during Exposure to Zooplankton.

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.

Diversifying selection by Desmodiinae legume species on Bradyrhizobium symbionts.

Desmodium and Hylodesmum (Papilionoideae Subtribe Desmodiinae) are among the most common herbaceous perennial legumes native to eastern North America. To analyze the population structure of their Bradyrhizobium sp. root-nodule bacteria, 159 isolates were sampled from ten host species across a 1000 km region. Phylogenetic analysis of four housekeeping loci (2164 bp) and two loci in the symbiosis island (SI) chromosomal region (1374 bp) indicated extensive overlap in symbiont utilization, with each common bacterial clade found on 2-7 species of these legume genera. However, host species differed considerably in the relative proportion of symbionts belonging to different Bradyrhizobium clades. High phylogenetic incongruence between trees for housekeeping loci and SI loci suggested that diversification of these Bradyrhizobium lineages involved substantial horizontal gene transfer. Plant inoculation with strains from six Bradyrhizobium clades revealed marked disparity in relative bacterial reproductive success across four Desmodium species. Estimated yield of Bradyrhizobium progeny cells per plant ranged from zero to >10(9), and strains with high fitness on one host sometimes reproduced poorly on other host species. Diversifying selection on bacteria, arising from differential success in habitats with different Desmodium and Hylodesmum taxa, is therefore likely to affect Bradyrhizobium diversity patterns at the landscape level.