Cyanoseq: A database of cyanobacterial 16S rRNA gene sequences with curated taxonomy.

Cyanobacteria are photosynthetic bacteria that occupy various habitats across the globe, playing critical roles in many of Earth's biogeochemical cycles both in both aquatic and terrestrial systems. Despite their well-known significance, their taxonomy remains problematic and is the subject of much research. Taxonomic issues of Cyanobacteria have consequently led to inaccurate curation within known reference databases, ultimately leading to problematic taxonomic assignment during diversity studies. Recent advances in sequencing technologies have increased our ability to characterize and understand microbial communities, leading to the generation of thousands of sequences that require taxonomic assignment. We herein propose CyanoSeq (https://zenodo.org/record/7569105), a database of cyanobacterial 16S rRNA gene sequences with curated taxonomy. The taxonomy of CyanoSeq is based on the current state of cyanobacterial taxonomy, with ranks from the domain to genus level. Files are provided for use with common naive Bayes taxonomic classifiers, such as those included in DADA2 or the QIIME2 platform. Additionally, FASTA files are provided for creation of de novo phylogenetic trees with (near) full-length 16S rRNA gene sequences to determine the phylogenetic relationship of cyanobacterial strains and/or ASV/OTUs. The database currently consists of 5410 cyanobacterial 16S rRNA gene sequences along with 123 Chloroplast, Bacterial, and Vampirovibrionia (formally Melainabacteria) sequences.

Novel diversity within Roseofilum (Desertifilaceae, Cyanobacteria) from marine benthic mats with description of four new species.

Benthic cyanobacterial mats (BCMs) are natural phenomena in marine environments. Reports of BCMs occurring across coastal marine environments have increased, partly driven by nutrient loading and climate change; thus, there is a need to understand the diversity involved in the proliferations and potential toxicity of the BCMs. Furthermore, marine cyanobacterial mats are observed growing on and affecting the health of corals with one specific cyanobacterial genus, Roseofilum, dominating the microbial mats associated with black band disease (BBD), a destructive polymicrobial disease that affects corals. To explore the diversity of Roseofilum, cyanobacterial mats from various marine habitats were sampled, and individual isolates were identified based on morphology, 16S rRNA gene phylogenies, 16S-23S ITS rRNA region sequence dissimilarities, and phylogenomics. Four novel species of Roseofilum were isolated from benthic marine mats, three from the coasts of Florida, United States (R. capinflatum sp. nov., R. casamattae sp. nov., and R. acuticapitatum sp. nov.) and one from the coast of France (R. halophilum sp. nov.). Our analyses revealed that Roseofilum associated with coral BBD and those not associated with corals but rather from coastal benthic mats are systematically distinct based on both phylogenetic and phylogenomic analyses. Enzyme-linked immunosorbent assay (ELISA) and LC-MS data indicated that microcystin production was found in one of the four species.

Untangling filamentous marine cyanobacterial diversity from the coast of South Florida with the description of Vermifilaceae fam. nov. and three new genera: Leptochromothrix gen. nov., Ophiophycus gen. nov., and Vermifilum gen. nov.

Benthic cyanobacterial mats are an integral component of aquatic communities in tropical marine waters. These mats can develop into massive nuisances at risk of expansion due to climate change. The extent of diversity occurring within these mats, still remains largely unexplored, especially in Florida. To reveal this diversity, coastal environments of South Florida were sampled and subsequently processed for isolation and systematic identification. Three new genera are proposed based on the molecular phylogeny, morphology, and ecology. These new genera are characterized by discoid cells and homocytous, unbranched filaments without sheaths. Individual genus morphological differences include either rounded bent, rounded, or conical rounded apical cells. A unique molecular fingerprint including a base pair insert within the 16S rRNA gene sequence and genetic similarities facilitates the delimitation of a novel family Vermifilaceae. Using the polyphasic approach, our research presents three new genera and four new species of marine cyanobacteria inhabiting coastal ecosystems of South Florida.

The occurrence of Affixifilum gen. nov. and Neolyngbya (Oscillatoriaceae) in South Florida (USA), with the description of A. floridanum sp. nov. and N. biscaynensis sp. nov.

South Florida (USA) has a subtropical to tropical climate with an extensive and diverse coastline that supports the growth of benthic cyanobacterial mats (BCMs). These BCMs are widespread and potentially house numerous bioactive compounds; however, the extent of the cyanobacterial diversity within these mats remains largely unknown. To elucidate this diversity, BCMs from select locations in South Florida were sampled and isolated into unicyanobacterial cultures for morphological and molecular studies. Phylogenetic relationships of isolated taxa were assessed using the markers 16S rRNA and 16S-23S rRNA ITS by both maximum likelihood and Bayesian inference. We propose Affixifilum gen. nov. based on morphological characteristics and the 16S rRNA phylogeny. Two species are included: Affixifilum granulosum comb nov. (=Neolyngbya granulosa) found in Brazil and Florida (USA) and A. floridanum sp. nov. Several other features, including pair-wise distance of 16S rRNA and 16S-23S rRNA ITS, 16S-23S rRNA ITS secondary structure, morphology, and ecology, provide support for Affixifilum. We also propose the transfer of Lyngbya regalis to Neolyngbya as N. regalis comb. nov. and include the description of one novel species, N. biscaynensis sp. nov.

Laboratory-scale evaluation of algaecide effectiveness for control of microcystin-producing cyanobacteria from Lake Okeechobee, Florida (USA).

Growth of microcystin-producing cyanobacteria in Lake Okeechobee (Florida, USA) and surrounding waters has resulted in adverse health impacts for humans and endangered species, as well as significant economic losses. As these issues worsen, there is growing pressure for efficacious solutions to rapidly mitigate harmful algal blooms (HABs) and protect critical freshwater resources. Applications of USEPA-registered algaecides as management tactics meet many decision-making criteria often required by water resource managers (e.g., effective, scalable, selective), but have not yet been evaluated on a large scale within the Lake Okeechobee waterway. This study was conducted to bolster the peer-reviewed database for available management tactics against microcystin-producing cyanobacteria in waters of this region. Laboratory-scale experiments can be conducted first to minimize uncertainty at larger scales and improve confidence in decision-making. In this study, samples containing microcystin-producing cyanobacteria collected from Lake Okeechobee were exposed to several USEPA-registered algaecides in laboratory toxicity experiments. Responses of target cyanobacteria were measured 3 days after treatment (DAT) in terms of cell density, chlorophyll-a concentrations, and phycocyanin concentrations. Based on responses of the cyanobacteria, minimum effective exposure concentrations were identified for each algaecide. Microcystin release (i.e. proportion of total microcystins in the aqueous phase) was measured and compared 1 DAT among effective exposures. Total microcystin concentrations were measured in effective treatments at 1, 4, and 9 DAT to discern potential for microcystin persistence following exposures to the effective formulations and exposure concentrations. Overall, several formulations including GreenClean Liquid® 5.0, GreenClean Liquid® 5.0 combined with Hydrothol® 191, and the copper-based algaecides evaluated (Algimycin® PWF, Argos, Captain® XTR, Cutrine® Ultra, and SeClear®) achieved significant and similar effects on target cyanobacteria. The chelated copper-based formulations (Algimycin® PWF, Argos, Captain® XTR, and Cutrine® Ultra) resulted in relatively less microcystin release 1 DAT and lesser total microcystin concentrations 4 DAT. At 9 DAT, total microcystin concentrations were significantly lower than in untreated controls in all treatments evaluated. These results provide the necessary comparative performance data for preliminary decision-making and designing additional studies at larger scales. Importantly, the comparative toxicity data and approach provided in this study demonstrate the initial steps for development of site-specific management strategies for Lake Okeechobee and other areas impacted by harmful algal blooms with large spatial and temporal scales.

Macrophyte coverage drives microbial community structure and interactions in a shallow sub-tropical lake.

Shallow lakes are typically dominated by macrophytes, which have important functional roles regulating trophic conditions and creating biological habitat. Macrophytes have been shown to strongly influence water chemistry and shape microbial communities in shallow lakes. In Florida, many large, shallow lakes are dominated by alien invasive, submersed macrophytes, such as hydrilla (Hydrilla verticillata [L.F.] Royle) and are intensively managed to reduce infestations and contain the spread of these alien invasive macrophytes. In this study, we investigated the effects of large (40 ha) herbicidal and mechanical control treatments on a large lake located in Central Florida that resulted in the reduction of Hydrilla and concomitant changes in water chemistry and microbial communities (both bacteria and protists [microbial eukaryotes]). We observed a considerable decrease in macrophyte coverage associated with plant control treatments as well as a temporal change in macrophyte coverage in Lake Tohopekaliga. We found that changes in macrophyte coverage, regardless of treatment type, significantly affected the water chemistry of the lake, resulting in a sharp increase of chlorophyll a concentration as well as an increase in turbidity with the decrease of macrophyte coverage. Moreover, the decline in macrophytes led to decreases in microbial community diversity with over-representation of phototrophic functional groups. Specifically, we observed an increase in cyanobacteria with the decrease in macrophyte coverage. Our study highlights the advantages and disadvantages of macrophyte control. Although there was an initial decrease in macrophyte coverage associated with the chemical and mechanical control of aquatic plants, after a few months, we found a considerable increase in coverage. In addition, the increase of cyanobacterial relative abundance demonstrates the possible consequences of aquatic plant control such as cyanobacterial blooms if there is a continued decline of macrophytes.

Microbial Community Response to Granular Peroxide-Based Algaecide Treatment of a Cyanobacterial Harmful Algal Bloom in Lake Okeechobee, Florida (USA).

Cyanobacterial harmful algal blooms (cyanoHABs) occur in fresh water globally. These can degrade water quality and produce toxins, resulting in ecological and economic damages. Thus, short-term management methods (i.e., algaecides) are necessary to rapidly mitigate the negative impacts of cyanoHABs. In this study, we assess the efficacy of a hydrogen peroxide-based algaecide (PAK® 27) on a Microcystis dominated bloom which occurred within the Pahokee Marina on Lake Okeechobee, Florida, USA. We observed a significant reduction in chlorophyll a (96.81%), phycocyanin (93.17%), and Microcystis cell counts (99.92%), and a substantial reduction in microcystins (86.7%) 48 h after treatment (HAT). Additionally, there was a significant shift in bacterial community structure 48 HAT, which coincided with an increase in the relative abundance of photosynthetic protists. These results indicate that hydrogen peroxide-based algaecides are an effective treatment method for cyanoHAB control and highlight their effects on non-target microorganisms (i.e., bacteria and protists).

Draft Genome Sequences of Bacteria with Cyanocidal Potential Isolated from Eutrophic Waters Associated with Cyanobacterial Harmful Algal Blooms.

Here, we report the draft genome sequences of nine bacterial species isolated from eutrophic waters associated with cyanobacterial harmful algal blooms with cyanocidal potential.

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.

Trophic State Drives the Diversity of Protists in a Tropical River (New River, Belize).

Land use disrupts the ecosystem functioning of freshwater systems and significantly affects trophic state. Consequently, biodiversity is severely affected by changes to the ecosystem. Microbial eukaryotes (i.e., protists) play an essential role in ecosystem functioning, contributing to biogeochemical processes, nutrient cycling, and food webs. Protist composition is a useful biological quality parameter for monitoring aquatic ecosystems and determining aquatic system health. In this study, we investigated the effects of land usage and trophic state on the communities of microbial eukaryotes in the New River (Belize, C.A.). Land use and trophic state both significantly affected protist community compositions, with impacted and mesotrophic sampled sites having higher biodiversity when compared to other sites. Autotrophic organisms dominated indirectly impacted and eutrophic sites, while impacted and mesotrophic sites had proportional ratios of autotrophic and heterotrophic organisms. Our study highlights the significant effects of trophic gradients on protistan community composition, even at the local scales.

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.

Genome Sequences of Two Microcystis aeruginosa (Chroococcales, Cyanobacteria) Strains from Florida (United States) with Disparate Toxigenic Potentials.

Here, we report the draft genomes of two Microcystis aeruginosa strains, i.e., M. aeruginosa BLCC-F108, which was isolated from a toxic bloom in eutrophic waters in Lake Okeechobee (Florida, USA), and M. aeruginosa BLCC-F158, which was isolated from mesotrophic waters in Lake Tohopekaliga (Florida, USA). Genomic analyses show disparate toxin potentials for these two strains.