Spatial and Temporal Resolution of Cyanobacterial Bloom Chemistry Reveals an Open-Ocean Trichodesmium thiebautii as a Talented Producer of Specialized Metabolites.

While the ecological role that Trichodesmium sp. play in nitrogen fixation has been widely studied, little information is available on potential specialized metabolites that are associated with blooms and standing stock Trichodesmium colonies. While a collection of biological material from a T. thiebautii bloom event from North Padre Island, Texas, in 2014 indicated that this species was a prolific producer of chlorinated specialized metabolites, additional spatial and temporal resolution was needed. We have completed these metabolite comparison studies, detailed in the current report, utilizing LC-MS/MS-based molecular networking to visualize and annotate the specialized metabolite composition of these Trichodesmium blooms and colonies in the Gulf of Mexico (GoM) and other waters. Our results showed that T. thiebautii blooms and colonies found in the GoM have a remarkably consistent specialized metabolome. Additionally, we isolated and characterized one new macrocyclic compound from T. thiebautii, trichothilone A (1), which was also detected in three independent cultures of T. erythraeum. Genome mining identified genes predicted to synthesize certain functional groups in the T. thiebautii metabolites. These results provoke intriguing questions of how these specialized metabolites affect Trichodesmium ecophysiology, symbioses with marine invertebrates, and niche development in the global oligotrophic ocean.

The Influence of Bacteria on the Growth, Lipid Production, and Extracellular Metabolite Accumulation by Phaeodactylum tricornutum (Bacillariophyceae).

To examine the impact of heterotrophic bacteria on microalgal physiology, we co-cultured the diatom Phaeodactylum tricornutum with six bacterial strains to quantify bacteria-mediated differences in algal biomass, total intracellular lipids, and for a subset, extracellular metabolite accumulation. A Marinobacter isolate significantly increased algal cell concentrations, dry biomass, and lipid content compared to axenic algal cultures. Two other bacterial strains from the Bacteroidetes order, of the genera Algoriphagus and Muricauda, significantly lowered P. tricornutum biomass, leading to overall decreased lipid accumulation. These three bacterial co-cultures (one mutualistic, two competitive) were analyzed for extracellular metabolites via untargeted liquid chromatography mass spectrometry to compare against bacteria-free cultures. Over 80% of the extracellular metabolites differentially abundant in at least one treatment were in higher concentrations in the axenic cultures, in agreement with the hypothesis that the co-cultured bacteria incorporated algal-derived organic compounds for growth. Furthermore, the extracellular metabolite profiles of the two growth-inhibiting cultures were more similar to one another than the growth-promoting co-culture, linking metabolite patterns to ecological role. Our results show that algal-bacterial interactions can influence the accumulation of intracellular lipids and extracellular metabolites, and suggest that utilization and accumulation of compounds outside the cell play a role in regulating microbial interactions.

Odorella benthonica gen. & sp. nov. (Pleurocapsales, Cyanobacteria): an odor and prolific toxin producer isolated from a California aqueduct.

Pleurocapsales are one of the least understood groups of cyanobacteria in terms of molecular systematics and biochemistry. Considering the high number of cryptic taxa within the Synechococcales and Oscillatoriales, it is likely that such taxa also occur in the Pleurocapsales. The new genus described in our research is the first known pleurocapsalean cryptic taxon. It produces off-flavor and a large number of bioactive metabolites (n = 38) some of which can be toxic including four known microcystins. Using a polyphasic approach, we propose the establishment of the genus Odorella with the new species O. benthonica from material originally isolated from the California Aqueduct near Los Angeles.

Cyanobufalins: Cardioactive Toxins from Cyanobacterial Blooms.

Cyanobufalins A-C (1-3), a new series of cardiotoxic steroids, have been discovered from cyanobacterial blooms in Buckeye Lake and Grand Lake St. Marys in Ohio. Compounds 1-3 contain distinctive structural features, including geminal methyl groups at C-4, a 7,8 double bond, and a C-16 chlorine substituent that distinguish them from plant- or animal-derived congeners. Despite these structural differences, the compounds are qualitatively identical to bufalin in their cytotoxic profiles versus cell lines in tissue culture and cardiac activity, as demonstrated in an impedance-based cellular assay conducted with IPSC-derived cardiomyocytes. Cyanobufalins are nonselectively toxic to human cells in the single-digit nanomolar range and show stimulation of contractility in cardiomyocytes at sub-nanomolar concentrations. The estimated combined concentration of 1-3 in the environment is in the same nanomolar range, and consequently more precise quantitative analyses are recommended along with more detailed cardiotoxicity studies. This is the first time that cardioactive steroid toxins have been found associated with microorganisms in an aquatic environment. Several factors point to a microbial biosynthetic origin for the cyanobufalins.

Ecophysiological Examination of the Lake Erie Microcystis Bloom in 2014: Linkages between Biology and the Water Supply Shutdown of Toledo, OH.

Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (U.S./Canada) during summer months since 1995. The production of toxins by bloom-forming cyanobacteria can lead to drinking water crises, such as the one experienced by the city of Toledo in August of 2014, when the city was rendered without drinking water for >2 days. It is important to understand the conditions and environmental cues that were driving this specific bloom to provide a scientific framework for management of future bloom events. To this end, samples were collected and metatranscriptomes generated coincident with the collection of environmental metrics for eight sites located in the western basin of Lake Erie, including a station proximal to the water intake for the city of Toledo. These data were used to generate a basin-wide ecophysiological fingerprint of Lake Erie Microcystis populations in August 2014 for comparison to previous bloom communities. Our observations and analyses indicate that, at the time of sample collection, Microcystis populations were under dual nitrogen (N) and phosphorus (P) stress, as genes involved in scavenging of these nutrients were being actively transcribed. Targeted analysis of urea transport and hydrolysis suggests a potentially important role for exogenous urea as a nitrogen source during the 2014 event. Finally, simulation data suggest a wind event caused microcystin-rich water from Maumee Bay to be transported east along the southern shoreline past the Toledo water intake. Coupled with a significant cyanophage infection, these results reveal that a combination of biological and environmental factors led to the disruption of the Toledo water supply. This scenario was not atypical of reoccurring Lake Erie blooms and thus may reoccur in the future.

Identification of a new-to-science cyanobacterium, Toxifilum mysidocida gen. nov. & sp. nov. (Cyanobacteria, Cyanophyceae).

Cyanobacteria occupy many niches within terrestrial, planktonic, and benthic habitats. The diversity of habitats colonized, similarity of morphology, and phenotypic plasticity all contribute to the difficulty of cyanobacterial identification. An unknown marine filamentous cyanobacterium was isolated from an aquatic animal rearing facility having mysid mortality events. The cyanobacterium originated from Corpus Christi Bay, TX. Filaments are rarely solitary, benthic mat forming, unbranched, and narrowing at the ends. Cells are 2.1 × 3.1 µm (width × length). Thylakoids are peripherally arranged on the outer third of the cell; cyanophycin granules and polyphosphate bodies are present. Molecular phylogenetic analysis in addition to morphology (transmission electron microscopy and scanning electron microscopy) and chemical composition all confirm it as a new genus and species we name Toxifilum mysidocida. At least one identified Leptolyngbya appears (based on genetic evidence and TEM) to belong to this new genus.

Trichothiazole A, a dichlorinated polyketide containing an embedded thiazole isolated from Trichodesmium blooms.

Mass spectrometry-guided isolation of the lipophilic extract of Trichodesmium bloom material led to the isolation and structure characterization of a new thiazole-containing di-chlorinated polyketide (1). The structure of 1 was deduced using 1D and 2D NMR analysis, high-resolution mass spectrometry analysis and complementary spectroscopic procedures. Trichothiazole A possesses interesting structural features, such as a terminal alkyne, two vinyl chlorides and a 2,4-disubstituted thiazole. Trichothiazole A showed moderate cytotoxicity to Neuro-2A cells (EC50: 13.3 ± 1.1 µM).

Trichophycin A, a Cytotoxic Linear Polyketide Isolated from a Trichodesmium thiebautii Bloom.

In an effort to isolate and characterize bioactive secondary metabolites from Trichodesmium thiebautii blooms, collected cyanobacteria biomass was subjected to bioassay-guided extraction and fractionation using the human colon cancer cell line HCT-116, resulting in the isolation and subsequent structure characterization of a linear polyketide trichophycin A (1). The planar structure of 1 was completed using 1D and 2D NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). Trichophycin A was moderately toxic against the murine neuroblastoma cell line Neuro-2A (EC50: 6.5 µM) and HCT-116 cells (EC50: 11.7 µM). Trichophycin A was significantly more cytotoxic than the previously isolated polyketides trichotoxin A and trichotoxin B. These cytotoxicity observations suggest that toxicity may be related to the polyol character of these polyketide compounds.

Tricholides A and B and Unnarmicin D: New Hybrid PKS-NRPS Macrocycles Isolated from an Environmental Collection of Trichodesmium thiebautii.

Bioassay-guided isolation of the lipophilic extract of Trichodesmium thiebautii bloom material led to the purification and structure characterization of two new hybrid polyketide-non-ribosomal peptide (PKS-NRPS) macrocyclic compounds, tricholides A and B (1 and 2). A third macrocyclic compound, unnarmicin D (3), was identified as a new depsipeptide in the unnarmicin family, given its structural similarity to the existing compounds in this group. The planar structures of 1-3 were determined using 1D and 2D NMR spectra and complementary spectroscopic and spectrometric procedures. The absolute configurations of the amino acid components of 1-3 were determined via acid hydrolysis, derivitization with Marfey's reagent and HPLC-UV comparison to authentic amino acid standards. The absolute configuration of the 3-hydroxydodecanoic acid moiety in 3 was determined using a modified Mosher's esterification procedure on a linear derivative of tricharmicin (4) and additionally by a comparison of 13C NMR shifts of 3 to known depsipeptides with ß-hydroxy acid subunits. Tricholide B (2) showed moderate cytotoxicity to Neuro-2A murine neuroblastoma cells (EC50: 14.5 ± 6.2 µM).

Structure revision of trichotoxin, a chlorinated polyketide isolated from a Trichodesmium thiebautii bloom.

NMR-guided fractionation of the lipophilic extract of Trichodesmium thiebautii filaments led to the isolation of a phenyl-containing chlorinated polyketide (1) and an alkyne-containing analogue (2). Comparison of spectroscopic and spectrometric data of 1 with the data of the previously reported trichotoxin, strongly suggested that these metabolites were identical and supports a structural revision of trichotoxin and its designation as trichotoxin A. In addition, we report the isolation and characterization of the alkyne-containing analogue trichotoxin B (2). Absolute configuration of 1 and 2 is proposed based on spectroscopic comparison to a close structural analog.

Benthic microalgae serve as the major food resource for porcelain crabs (Petrolisthes spp.) in oyster reefs: gut content and pigment evidence1.

Suspension-feeding porcelain crabs (Petrolisthes spp.) are often the most abundant decapod crustaceans in oyster reef habitat. Analysis of water column and subtidal algal biomass from three Texas estuaries suggests that planktonic food resources are insufficient for porcelain crab growth. Pigment composition of porcelain crab muscle and digestive track contents included the diatom pigment fucoxanthin and cyanobacterial pigment canthaxanthin with digestive track samples containing attached (adnate) benthic diatoms as well as benthic cyanobacteria not found in the water column. Feeding appendages on porcelain crabs include numerous cirri with serrated edges as well as fewer more brush-like longer units. Benthic food resources are in sufficient supply to support porcelain crab biomass.

Augmenting anti-cancer natural products with a small molecule adjuvant.

Aquatic microbes produce diverse secondary metabolites with interesting biological activities. Cytotoxic metabolites have the potential to become lead compounds or drugs for cancer treatment. Many cytotoxic compounds, however, show undesirable toxicity at higher concentrations. Such undesirable activity may be reduced or eliminated by using lower doses of the cytotoxic compound in combination with another compound that modulates its activity. Here, we have examined the cytotoxicity of four microbial metabolites [ethyl N-(2-phenethyl) carbamate (NP-1), Euglenophycin, Anabaenopeptin, and Glycolipid 652] using three in vitro cell lines [human breast cancer cells (MCF-7), mouse neuroblastoma cells (N2a), and rat pituitary epithelial cells (GH4C1)]. The compounds showed variable cytotoxicity, with Euglenophycin displaying specificity for N2a cells. We have also examined the modulatory power of NP-1 on the cytotoxicity of the other three compounds and found that at a permissible concentration (125 µg/mL), NP-1 sensitized N2a and MCF-7 cells to Euglenophycin and Glycolipid 652 induced cytotoxicity.

Role of serotonin in the lack of sensitization caused by prolonged food deprivation in Aplysia.

Food deprivation may cause neurological dysfunctions including memory impairment. The mollusk Aplysia is a suitable animal model to study prolonged food deprivation-induced memory deficits because it can sustain up to 14 days of food deprivation (14DFD). Sensitization of defensive withdrawal reflexes has been used to illustrate the detrimental effects of 14DFD on memory formation. Under normal feeding conditions (i.e., two days food deprivation, 2DFD), aversive stimuli lead to serotonin (5-HT) release into the hemolymph and neuropil, which mediates sensitization and its cellular correlates including increased excitability of tail sensory neurons (TSNs). Recent studies found that 14DFD prevents both short-term and long-term sensitization, as well as short-term increased excitability of TSNs induced by in vitro aversive training. This study investigated the role of 5-HT in the absence of sensitization and TSN increased excitability under 14DFD. Because 5-HT is synthesized from tryptophan obtained through diet, and its exogeneous application alone induces sensitization and increases TSN excitability, we hypothesized that 1) 5-HT level may be reduced by 14DFD and 2) 5-HT may still induce sensitization and TSN increased excitability in 14DFD animals. Results revealed that 14DFD significantly decreased hemolymph 5-HT level, which may contribute to the lack of sensitization and its cellular correlates, while ganglia 5-HT level was not changed. 5-HT exogenous application induced sensitization in 14DFD Aplysia, albeit smaller than that in 2DFD animals, suggesting that this treatment can only induce partial sensitization in food deprived animals. Under 14DFD, 5-HT increased TSN excitability indistinguishable from that observed under 2DFD. Taken together, these findings characterize 5-HT metabolic deficiency under 14DFD, which may be compensated, at least in part, by 5-HT exogenous application.

Resolving mixed algal species in hyperspectral images.

We investigated a lab-based hyperspectral imaging system's response from pure (single) and mixed (two) algal cultures containing known algae types and volumetric combinations to characterize the system's performance. The spectral response to volumetric changes in single and combinations of algal mixtures with known ratios were tested. Constrained linear spectral unmixing was applied to extract the algal content of the mixtures based on abundances that produced the lowest root mean square error. Percent prediction error was computed as the difference between actual percent volumetric content and abundances at minimum RMS error. Best prediction errors were computed as 0.4%, 0.4% and 6.3% for the mixed spectra from three independent experiments. The worst prediction errors were found as 5.6%, 5.4% and 13.4% for the same order of experiments. Additionally, Beer-Lambert's law was utilized to relate transmittance to different volumes of pure algal suspensions demonstrating linear logarithmic trends for optical property measurements.

Uptake of Nanoplastic particles by zebrafish embryos triggers the macrophage response at early developmental stage.

Plastic pollution continues to erupt as a global ecological concern. As plastic debris is degraded into nanoscale and microscale particles via biodegradation, UV-irradiation, and mechanical processes, nanoplastic pollution arises as a threat to virtually every biological and ecological system on the planet. In this study, zebrafish (Danio rerio) embryos were exposed to fluorescently labeled plastic particles at nanoscales (30 nm and 100 nm). The uptake of both the nanoplastic particles (NPs) was found to exponentially increase with incubation time. Penetration of NPs through the natural barrier of the zebrafish embryos, the chorion, was observed prior to the hatching of the embryo. As a result, the NPs were found to accumulate on the body surface as well as inside the body of the zebrafish. The invasion of NPs into zebrafish embryos induced the upregulation of several stress and immune response genes including interleukins (il6 and il1b), cytochrome P450 (cyp1a and cyp51), and reactive oxygen species (ROS) removal protein-encoding genes (sod and cat). This suggested the initiation of ROS generation and removal as well as the activation of the immune response of zebrafish embryos. Colocalization of macrophages and NPs in zebrafish embryos indicated the involvement of macrophage response to the NP invasion at the early developmental stage of zebrafish.

Spatiotemporal diversity and community structure of cyanobacteria and associated bacteria in the large shallow subtropical Lake Okeechobee (Florida, United States).

Lake Okeechobee is a large eutrophic, shallow, subtropical lake in south Florida, United States. Due to decades of nutrient loading and phosphorus rich sediments, the lake is eutrophic and frequently experiences cyanobacterial harmful algal blooms (cyanoHABs). In the past, surveys of the phytoplankton community structure in the lake have been conducted by morphological studies, whereas molecular based studies have been seldom employed. With increased frequency of cyanoHABs in Lake Okeechobee (e.g., 2016 and 2018 Microcystis-dominated blooms), it is imperative to determine the diversity of cyanobacterial taxa that exist within the lake and the limnological parameters that drive bloom-forming genera. A spatiotemporal study of the lake was conducted over the course of 1 year to characterize the (cyano)bacterial community structure, using 16S rRNA metabarcoding, with coincident collection of limnological parameters (e.g., nutrients, water temperature, major ions), and cyanotoxins. The objectives of this study were to elucidate spatiotemporal trends of community structure, identify drivers of community structure, and examine cyanobacteria-bacterial relationships within the lake. Results indicated that cyanobacterial communities within the lake were significantly different between the wet and dry season, but not between periods of nitrogen limitation and co-nutrient limitation. Throughout the year, the lake was primarily dominated by the picocyanobacterium Cyanobium. The bloom-forming genera Cuspidothrix, Dolichospermum, Microcystis, and Raphidiopsis were highly abundant throughout the lake and had disparate nutrient requirements and niches within the lake. Anatoxin-a, microcystins, and nodularins were detected throughout the lake across both seasons. There were no correlated (cyano)bacteria shared between the common bloom-forming cyanobacteria Dolichospermum, Microcystis, and Raphidiopsis. This study is the first of its kind to use molecular based methods to assess the cyanobacterial community structure within the lake. These data greatly improve our understanding of the cyanobacterial community structure within the lake and the physiochemical parameters which may drive the bloom-forming taxa within Lake Okeechobee.

Testing an Iron Oxide Nanoparticle-Based Method for Magnetic Separation of Nanoplastics and Microplastics from Water.

Nanoplastic pollution is increasing worldwide and poses a threat to humans, animals, and ecological systems. High-throughput, reliable methods for the isolation and separation of NMPs from drinking water, wastewater, or environmental bodies of water are of interest. We investigated iron oxide nanoparticles (IONPs) with hydrophobic coatings to magnetize plastic particulate waste for removal. We produced and tested IONPs synthesized using air-free conditions and in atmospheric air, coated with several polydimethylsiloxane (PDMS)-based hydrophobic coatings. Particles were characterized with scanning electron microscopy (SEM), transmission electron microscopy (TEM), superconducting quantum interference device (SQUID) magnetometry, dynamic light scattering (DLS), X-ray diffraction (XRD) and zeta potential. The IONPs synthesized in air contained a higher percentage of the magnetic spinel phase and stronger magnetization. Binding and recovery of NMPs from both salt and freshwater samples was demonstrated. Specifically, we were able to remove 100% of particles in a range of sizes, from 2-5 mm, and nearly 90% of nanoplastic particles with a size range from 100 nm to 1000 nm using a simple 2-inch permanent NdFeB magnet. Magnetization of NMPs using IONPs is a viable method for separation from water samples for quantification, characterization, and purification and remediation of water.

Euglena International Network (EIN): Driving euglenoid biotechnology for the benefit of a challenged world.

Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.

Diversity of bioactive compound content across 71 genera of marine, freshwater, and terrestrial cyanobacteria.

Cyanobacterial blooms have increased in frequency, distribution, and intensity due to climate change and anthropogenic nutrient input. The release of bioactive compounds accumulated in these blooms can affect the health of humans and the environment. The co-occurrence of bioactive metabolites is well-documented in bloom samples from marine and freshwater ecosystems, with fewer reports from unialgal isolates. Cyanobacteria also are important terrestrial ecosystem components, especially in drylands, but reports on bioactive molecules from terrestrial cyanobacteria are sparse. This study determined bioactive metabolite profiles for 71 genera of cyanobacteria from seven orders isolated from freshwater (12 genera), marine (15 genera), and terrestrial (44 genera) habitats originally. Cultures were harvested for bioactive metabolites when entering the late-exponential phase for all 157 strains, and 33 were sampled at both early and late exponential phases. Bioactive metabolites were analyzed using an ultra high performance/pressure liquid chromatography in-line with a time-of-flight mass spectrometer. Overall, 12 bioactive classes of the 28 identified were ubiquitous in all samples. On average, each freshwater genus produced ∼12 bioactive classes, whereas each marine genus contained > 4 bioactive classes, and each terrestrial genus contained ∼6 bioactive classes. While 10 of 12 freshwater genera produced at least 10 bioactive classes, only a single genus each from marine and terrestrial habitats had the same number of bioactive classed accumulated. Aeruginosin was found in 58 of 71 total genera, carmabin in 51 of 71 genera, and anabaenopeptin in 48 of 71 genera. Chemotaxonomic use of these secondary metabolites may help resolve higher-level genetic classification(s). An additional growth curve experiment showed that bioactive metabolites were produced at both early and late exponential growth phases. The bioactive metabolite accumulation pattern between early and late exponential phases differed by bioactive classes, genera, and habitats. This survey of 55 bioactive classes in cyanobacteria isolated from freshwater, marine, and terrestrial habitats (71 genera) provides as one of the first systematic bioactive metabolite profiles for cyanobacteria, which should be useful in environmental and drinking water management. Further, it offers novel insights about the toxin potential of selected terrestrial cyanobacteria.

Iningainema tapete sp. nov. (Scytonemataceae, Cyanobacteria) from greenhouses in central Florida (USA) produces two types of nodularin with biosynthetic potential for microcystin-LR and anabaenopeptin production.

Iningainema is a recently described genus of heterocytous, false-branching cyanobacteria originally described from Australia. In this work, we present Iningainema tapete sp. nov., isolated from subaerial and terrestrial environments in central Florida (USA). In comparison to the sister species, our novel cyanobacterium produces nodularin-R (NOD-R) and a methylated isoform [MeAdda3] NOD previously not reported within this genus; in addition to possessing the biosynthetic gene clusters for microcystin and anabaenopeptins production. Nodularin accumulation by this cyanobacterium exceeded 500 µg g-1 dry weight in cultures grown in nitrogen-depleted media. Such elevated toxin concentrations are alarming as the cyanobacterium was isolated from a food production greenhouse and poses a potential risk for food products and for workforce exposure. Using morphology, 16S rRNA gene phylogeny, and 16S-23S rRNA internal transcribed spacer (ITS) secondary structure, coupled with toxin detection and toxin gene presence, we provide evidence for the establishment of a novel toxic species of cyanobacteria, Iningainema tapete.