Intensification of harmful cyanobacterial blooms in a eutrophic, temperate lake caused by nitrogen, temperature, and CO2.

Warmer temperatures can significantly increase the intensity of cyanobacterial harmful algal blooms (CHABs) in eutrophic freshwater ecosystems. However, few studies have examined the effects of CO2 enrichment in tandem with elevated temperature and/or nutrients on cyanobacterial taxa in freshwater ecosystems. Here, we observed changes in the biomass of cyanobacteria, nutrients, pH, and carbonate chemistry over a two-year period in a shallow, eutrophic freshwater lake and performed experiments to examine the effects and co-effects of CO2, temperature, and nutrient enrichment on cyanobacterial and N2-fixing (diazotrophic) communities assessed via high throughput sequencing of the 16S rRNA and nifH genes, respectively. During both years, there were significant CHABs (50-500 µg cyanobacterial chlorophyll-a L-1) and lake CO2 levels were undersaturated (≤300 µatm pCO2). NH4+ significantly increased the net growth rates of cyanobacteria as well as the biomass of the diazotrophic cyanobacterial order Nostocales under elevated and ambient CO2 conditions. In a fall experiment, the N2 fixation rates of Nostocales were significantly higher when populations were enriched with CO2 and P, relative to CO2-enriched populations that were not amended with P. During a summer experiment, N2 fixation rates increased significantly under N and CO2 - enriched conditions relative to N-enriched and ambient CO2 conditions. Nostocales dominated the diazotrophic communities of both experiments, achieving the highest relative abundance under CO2-enriched conditions when N was added in the first experiment and when CO2 and temperature were elevated in the second experiment, when N2 fixation rates also increased significantly. Collectively, this study indicates that N promotes cyanobacterial blooms including those formed by Dolichospermum and that the biomass and N2 fixation rates of diazotrophic cyanobacterial taxa may benefit from enhanced CO2 levels in eutrophic lakes.

Elevated CO2 significantly increases N2 fixation, growth rates, and alters microcystin, anatoxin, and saxitoxin cell quotas in strains of the bloom-forming cyanobacteria, Dolichospermum.

The effect of rising CO2 levels on cyanobacterial harmful algal blooms (CHABs) is an emerging concern, particularly within eutrophic ecosystems. While elevated pCO2 has been associated with enhanced growth rates of some cyanobacteria, few studies have explored the effect of CO2 and nitrogen availability on diazotrophic (N2-fixing) cyanobacteria that produce cyanotoxins. Here, the effects of elevated CO2 and fixed nitrogen (NO3-) availability on the growth rates, toxin production, and N2 fixation of microcystin, saxitoxin, and anatoxin-a - producing strains of the genus Dolichospermum were quantified. Growth rates of all Dolichospermum spp. were significantly increased by CO2 or both CO2 and NO3- with rates being highest in treatments with the highest levels of CO2 and NO3-for all strains. While NO3- suppressed N2 fixation, diazotrophy significantly increased when NO3--enriched Dolichospermum spp. were supplied with higher CO2 compared to cultures grown under lower CO2 levels. This suggests that diazotrophy will play an increasingly important role in N cycling in CO2-enriched, eutrophic lentic systems. NO3- significantly increased quotas of the N-rich cyanotoxins, microcystin and saxitoxin, at ambient and enriched CO2 levels, respectively. In contrast, elevated CO2 significantly decreased cell quotas of microcystin and saxitoxin, but significantly increased cell quotas of the N-poor cyanotoxin, anatoxin. N2 fixation was significantly negatively and positively correlated with quotas of N-rich and N-poor cyanotoxins, respectively. Findings suggest cellular quotas of N-rich toxins (microcystin and saxitoxin) may be significantly reduced, or cellular quotas of N-poor toxins (anatoxin) may be significantly enhanced, under elevated CO2 conditions during diazotrophic cyanobacterial blooms. Finally, in the future, ecosystems that experience combinations of excessive N loading and CO2 enrichment may become more prone to toxic blooms of Dolichospermum.

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.

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.

Nitrogen limitation, toxin synthesis potential, and toxicity of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida, during the 2016 state of emergency event.

Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a state of emergency. This study reports on measurements and nutrient amendment experiments performed in this freshwater-estuarine ecosystem (salinity 0-25 PSU) during and after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-phosphorus ratios < 6, while Microcystis dominated (> 95%) phytoplankton inventories from the lake to the central part of the estuary. Chlorophyll a and microcystin concentrations peaked (100 and 34 µg L-1, respectively) within Lake Okeechobee and decreased eastwards. Metagenomic analyses indicated that genes associated with the production of microcystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originated from Microcystis and multiple diazotrophic genera, respectively. There were highly significant correlations between levels of total nitrogen, microcystin, and microcystin synthesis gene abundance across all surveyed sites (p < 0.001), suggesting high levels of nitrogen supported the production of microcystin during this event. Consistent with this, experiments performed with low salinity water from the St. Lucie River during the event indicated that algal biomass was nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups displayed nitrogen-limitation. These results indicate that monitoring and regulatory strategies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.

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.