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.

Transcriptomic and isotopic data reveal central role of ammonium in facilitating the growth of the mixotrophic dinoflagellate, Dinophysis acuminata.

Dinophysis spp. are mixotrophs that are dependent on specific prey, but are also potentially reliant on dissolved nutrients. The extent to which Dinophysis relies on exogenous N and the specific biochemical pathways important for supporting its autotrophic and heterotrophic growth are unknown. Here, the nutritional ecology of Dinophysis was explored using two approaches: 1) 15N tracer experiments were conducted to quantify the concentration-dependent uptake rates and associated kinetics of various N compounds (nitrate, ammonium, urea) of Dinophysis cultures and 2) the transcriptomic responses of Dinophysis cultures grown with multiple combinations of prey and nutrients were assessed via dinoflagellate spliced leader-based transcriptome profiling. Of the N compounds examined, ammonium had the highest Vmax and affinity coefficient, and lowest Ks for both pre-starved and pre-fed cultures, collectively demonstrating the preference of Dinophysis for this N source while little-to-no nitrate uptake was observed. During the transcriptome experiments, Dinophysis grown with nitrate and without prey had the largest number of genes with lower transcript abundances, did not increase abundance of transcripts associated with nitrate/nitrite uptake or reduction, and displayed no cellular growth, suggesting D. acuminata is not capable of growing on nitrate. When offered prey, the transcriptomic response of Dinophysis included the production of phagolysosomes, enzymes involved in protein and lipid catabolism, and N acquisition through amino acid degradation pathways. Compared with cultures only offered ammonium or prey, cultures offered both ammonium and prey had the largest number of genes with increased transcript abundances, the highest growth rate, and the unique activation of multiple pathways involved in cellular catabolism, further evidencing the ability of Dinophysis to grow optimally as a mixotroph. Collectively, this study evidences the key role ammonium plays in the mixotrophic growth of Dinophysis and reveals the precise biochemical pathways that facilitate its mixotrophic growth.

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.

Nitrogenous nutrients promote the growth and toxicity of Dinophysis acuminata during estuarine bloom events.

Diarrhetic Shellfish Poisoning (DSP) is a globally significant human health syndrome most commonly caused by dinoflagellates within the genus Dinophysis. While blooms of harmful algae have frequently been linked to excessive nutrient loading, Dinophysis is a mixotrophic alga whose growth is typically associated with prey availability. Consequently, field studies of Dinophysis and nutrients have been rare. Here, the temporal dynamics of Dinophysis acuminata blooms, DSP toxins, and nutrients (nitrate, ammonium, phosphate, silicate, organic compounds) were examined over four years within two New York estuaries (Meetinghouse Creek and Northport Bay). Further, changes in the abundance and toxicity of D. acuminata were assessed during a series of nutrient amendment experiments performed over a three year period. During the study, Dinophysis acuminata blooms exceeding one million cells L-1 were observed in both estuaries. Highly significant (p<0.001) forward stepwise multivariate regression models of ecosystem observations demonstrated that D. acuminata abundances were positively dependent on multiple environmental parameters including ammonium (p = 0.007) while cellular toxin content was positively dependent on ammonium (p = 0.002) but negatively dependent on nitrate (p<0.001). Nitrogen- (N) and phosphorus- (P) containing inorganic and organic nutrients significantly enhanced D. acuminata densities in nearly all (13 of 14) experiments performed. Ammonium significantly increased cell densities in 10 of 11 experiments, while glutamine significantly enhanced cellular DSP content in 4 of 5 experiments examining this compound. Nutrients may have directly or indirectly enhanced D. acuminata abundances as densities of this mixotroph during experiments were significantly correlated with multiple members of the planktonic community (phytoflagellates and Mesodinium). Collectively, this study demonstrates that nutrient loading and more specifically N-loading promotes the growth and toxicity of D. acuminata populations in coastal zones.

Shifts in Cyanobacterial Strain Dominance during the Onset of Harmful Algal Blooms in Florida Bay, USA.

Cyanobacteria are fundamental components of aquatic phytoplankton communities and some taxa can cause harmful blooms in coastal ecosystems. Harmful cyanobacterial blooms are typically comprised of multiple strains of a single genus or species that cannot be resolved microscopically. Florida Bay, USA, has experienced harmful cyanobacterial blooms that have been associated with the loss of eelgrass, spiny lobsters, and general food web disruption for more than two decades. To identify the strain or strains of cyanobacteria forming blooms in Florida Bay, samples were collected across the system over an annual cycle and analyzed via DNA sequencing using cyanobacterial-specific 16S rRNA gene primers, flow cytometry, and scanning electron microscopy. Analyses demonstrated that the onset of blooms in Florida Bay was coincident with a transformation of the cyanobacterial populations. When blooms were absent, the cyanobacterial population in Florida Bay was dominated by phycoerythrin-containing Synechococcus cells that were most similar to strains within Clade III. As blooms developed, the cyanobacterial community transitioned to dominance by phycocyanin-containing Synechococcus cells that were coated with mucilage, chain-forming, and genetically most similar to the coastal strains within Clade VIII. Clade VIII strains of Synechococcus are known to grow rapidly, utilize organic nutrients, and resist top-down control by protozoan grazers and viruses, all characteristics consistent with observations of cyanobacterial blooms in Florida Bay. Further, the strains of Synechococcus blooming in this system are genetically distinct from the species previously thought to cause blooms in Florida Bay, Synechococcus elongatus. Collectively, this study identified the causative organism of harmful cyanobacterial blooms in Florida Bay, demonstrates the dynamic nature of cyanobacterial stains within genera in an estuary, and affirms factors promoting Synechococcus blooms.

The effects of elevated CO2 on the growth and toxicity of field populations and cultures of the saxitoxin-producing dinoflagellate, Alexandrium fundyense.

The effects of coastal acidification on the growth and toxicity of the saxitoxin-producing dinoflagellate Alexandrium fundyense were examined in culture and ecosystem studies. In culture experiments, Alexandrium strains isolated from Northport Bay NY, USA, and the Bay of Fundy, Canada, grew significantly faster (16 -190%; p<0.05) when exposed to elevated levels of pCO2 (~ 800- 1900µatm) compared to lower levels (~390µatm). Exposure to higher levels of pCO2 also resulted in significant increases (71 - 81%) in total cellular toxicity (fg STX eq. cell-1) in the Northport Bay strain, while no changes in toxicity were detected in the Bay of Fundy strain. The positive relationship between pCO2 enhancement and elevated growth was reproducible using natural populations from Northport; Alexandrium densities were significantly and consistently enhanced when natural populations were incubated at 1500 µatm pCO2, a value at the upper range of those recorded in Northport Bay, 390 - 1500 µatm. During natural Alexandrium blooms in Northport Bay, pCO2 concentrations increased over the course of a bloom to more than 1700µatm and were highest in regions with the greatest Alexandrium abundances, suggesting Alexandrium may be further exacerbating acidification or be especially adapted to these extreme, acidified conditions. The co-occurrence of Alexandrium blooms and elevated pCO2 represents a previously unrecognized, compounding environmental threat to coastal ecosystems. The ability of elevated pCO2 to enhance the growth and toxicity of Alexandrium indicates that acidification promoted by eutrophication or climate change can intensify these, and perhaps other, harmful algal blooms.

Vitamin b(1) and b(12) uptake and cycling by plankton communities in coastal ecosystems.

While vitamin B(12) has recently been shown to co-limit the growth of coastal phytoplankton assemblages, the cycling of B-vitamins in coastal ecosystems is poorly understood as planktonic uptake rates of vitamins B(1) and B(12) have never been quantified in tandem in any aquatic ecosystem. The goal of this study was to establish the relationships between plankton community composition, carbon fixation, and B-vitamin assimilation in two contrasting estuarine systems. We show that, although B-vitamin concentrations were low (pM), vitamin concentrations and uptake rates were higher within a more eutrophic estuary and that vitamin B(12) uptake rates were significantly correlated with rates of primary production. Eutrophic sites hosted larger bacterial and picoplankton abundances with larger carbon normalized vitamin uptake rates. Although the >2 µm phytoplankton biomass was often dominated by groups with a high incidence of vitamin auxotrophy (dinoflagellates and diatoms), picoplankton (<2 µm) were always responsible for the majority of B(12)-vitamin uptake. Multiple lines of evidence suggest that heterotrophic bacteria were the primary users of vitamins among the picoplankton during this study. Nutrient/vitamin amendment experiments demonstrated that, in the Summer and Fall, vitamin B(12) occasionally limited or co-limited the accumulation of phytoplankton biomass together with nitrogen. Combined with prior studies, these findings suggest that picoplankton are the primary producers and users of B-vitamins in some coastal ecosystems and that rapid uptake of B-vitamins by heterotrophic bacteria may sometimes deprive larger phytoplankton of these micronutrients and thus influence phytoplankton species succession.