The Ecology of Benthic Toxigenic Anabaena and Phormidium (Cyanobacteria) in the Eel River, California

Cyanobacteria are ubiquitous in aquatic ecosystems across the earth. In many environments they are present at low abundances, however under certain environmental conditions cyanobacteria bloom and become one of the dominant organisms in an waterbody, degrading aquatic food webs and water quality. Cyanobacteria evolved over 2 billion years ago, and cyanobacterial harmful algal blooms (cyanoHABs) have been documented for decades. Of particular concern is the production of cyanotoxins, secondary metabolites toxic to humans and other organisms, by certain strains of cyanobacteria. Most research of cyanoHABs has been of planktonic blooms in lakes or estuaries, and cyanotoxin production by benthic cyanobacteria in rivers has been more recent, but in many rivers benthic cyanobacteria are the primary source of cyanotoxins. With field surveys and monitoring, manipulative field experiments, and genome-resolved metagenomics, this dissertation investigated the ecology of benthic cyanobacteria in the Eel River, California./nLike most coastal rivers in Northern California, the Eel River is in a Mediterranean climate. During the seasonal summer drought rivers become shallow, slow flowing, and warm, excellent habitat for benthic algal production. Non-toxic benthic algae are consumed by vertebrate and invertebrate grazers and are foundational to the aquatic summer food web. However, these benthic algal assemblages can tip towards toxicity when cyanobacteria begin to dominate the assemblage. Over a dozen dogs have died in the Eel River since the year 2000 due to ingesting cyanobacteria. Neighboring watersheds have also experienced dog deaths from cyanotoxin poisoning, and benthic cyanobacterial mats are an increasing public health concern in multiple Northern California rivers. Prior to this dissertation there were few data on the distribution and ecology of benthic toxigenic cyanobacteria in California rivers./nDuring the summers of 2013-2015, I documented spatial and temporal patterns of cyanobacterial occurrence and cyanotoxin concentrations in the watershed, showing widespread distribution of anatoxin-a produced by benthic cyanobacteria. Solid phase adsorption toxin tracking (SPATT) samplers were deployed weekly to record dissolved microcystin and anatoxin-a levels at 10 sites throughout the watershed, and 187 cyanobacterial mat samples were collected from 31 sites to measure intracellular anatoxin-a and microcystins. Anatoxin-a levels were higher than microcystin for both SPATT and cyanobacterial mat samples. Species of benthic Anabaena and Phormidium were frequently found to produce cyanotoxins in the Eel watershed. Of the benthic mats sampled, 50% had detectable anatoxin-a (mean µg g-1 DW= 1.71, max= 70.93), while 24% had detectable microcystins (mean µg g-1 DW= 0.067, max= 2.29). SPATT cyanotoxin levels peaked in mid-summer in warm mainstem reaches of the watershed. This is one of the first documentations of widespread anatoxin-a occurrence and anatoxin-a production by benthic cyanobacterial mats in a North American watershed./nField experiments were used to study the buoyancy of benthic Anabaena spp. mats to understand implications for Anabaena dispersal. Experiments addressed oxygen bubble production and dissolution on the buoyancy of Anabaena dominated benthic mats in response to light exposure. Samples of Anabaena dominated mats were harvested from the South Fork Eel River and placed in settling columns to measure floating and sinking velocities, or deployed into in situ ambient and low light treatments to measure the effect of light on flotation. Floating and sinking occurred within minutes and were driven by oxygen bubbles produced during photosynthesis, rather than intracellular changes in carbohydrates or gas vesicles. Light experiment results showed that in a natural ambient light regime, mats remained floating for at least 4 days, while in low light mats begin to sink in <24 hours. The ability of Anabaena mats to maintain their buoyancy will markedly increase their downstream dispersal distances. Increased buoyancy also allows toxin-containing mats to collect along shorelines, increasing threats to human and animal public health./nWithin cyanobacterial mats are a consortia of microbes interacting together to process and exchange molecules to maintain their growth. Currently, little is known about the diversity of the biosynthetic capacities of cyanobacterial species and associated microbes in freshwater benthic mats in rivers. I sampled 22 Phormidium mats collected across the Eel River network and used genome-resolved metagenomics to 1) investigate cyanobacterial and co-occurring microbial assemblage diversity, 2) probe their metabolic potential, and 3) evaluate their capacities for toxin production. From the genomes of seven strains from one species group we describe the first anatoxin-a operon from the genus Phormidium. Importantly, community composition within the mat appears to be associated with the presence of cyanobacteria capable of producing anatoxin-a. Bacteroidetes, Proteobacteria, and novel Verrucomicrobia dominated the microbial assemblages. Interestingly, some mats also contained Candidate Phylum Kapabacteria and Candidate Phyla Radiation bacteria from Absconditabacteria (SR1), Parcubacteria (OD1) and Doudnabacteria (SM2F11). Although the majority of genomes were unique to a particular sample, metabolic diversity was low across samples. In addition to oxygenic photosynthesis and carbon respiration, metabolic capacities include aerobic anoxygenic photosynthesis, sulfur compound oxidation and breakdown of urea. These results show the importance of organic carbon and nitrogen to energy flow and nutrient cycling within mats and the interactions between potential anatoxin-a production and microbial assemblage composition./nBy combining watershed-scale observations, field experiments, and genomic analyses this dissertation provides novel information about the ecology of toxigenic benthic cyanobacteria in California rivers. Comprehensive knowledge of the ecology of benthic cyanobacteria is necessary to understand it’s impacts to public and ecosystem health and to better predict where and when blooms might occur in rivers.

Advisor: Power, Mary E