Toxic benthic cyanobacteria (Microcoleus autumnalis): Genetic structure and ecological effects
Authors:   , Kelly Laura 
Published: December 2021

<p>Toxic, benthic cyanobacterial proliferations occur in rivers globally and are increasing in both frequency and severity. More than 100 dog deaths have been reported in New Zealand since 2005, and dogs have died due to cyanotoxins from benthic cyanobacteria in California and France in recent years. In New Zealand, toxic, benthic proliferations are typically dominated by the genus Microcoleus, which produces the potent neurotoxins called anatoxins. Investigations of the drivers of Microcoleus proliferations have been carried out, revealing important relationships between growth, nutrients and flow. Despite increased attention over the past 15 years, significant knowledge gaps remain regarding regulation of anatoxin concentrations in mats and the ecological impacts of toxic Microcoleus-dominated proliferations in aquatic ecosystems. In this study, I aimed to address these crucial knowledge gaps.  Concentrations of anatoxins in Microcoleus-dominated mats are highly variable, ranging from 0 μg g⁻¹ to over 2000 μg g⁻¹ dry weight. Like other cyanobacteria, both toxic and non-toxic strains coexist, and it is possible that the relative abundance of these strains is related to the observed variability in anatoxin concentrations. I developed a quantitative PCR assay specific to the Microcoleus anaC gene and combined this with a 16S rRNA quantitative PCR specific to cyanobacteria. This assay enabled the relative abundance of toxic and non-toxic strains to be quantified for the first time in environmental samples of Microcoleus-dominated mats. The quantitative PCR assay was applied to 122 environmental samples. Toxic cells made up 0% to 30.3% of the samples and significant differences were detectable among rivers, with wide variation evident both within and among rivers. Anatoxin contents in samples were significantly correlated with the proportion of toxic cells, suggesting that variation in anatoxin concentrations are related primarily to the dominance of toxic strains.   A field study was conducted in California to identify cyanotoxin producers, investigate the utility of the primers from the quantitative PCR developed in the previous chapter in an international setting and determine the extent of spatial variability in anatoxin concentrations and quotas. Samples were collected from the Eel and Russian rivers in northern California and gene screening, followed by sequencing, was conducted to identify likely cyanotoxin producers. Cyanotoxin concentrations were measured using liquid chromatography tandem-mass spectrometry and the anatoxin quotas were determined using droplet digital PCR. Cyanotoxin producers were identified as Microcoleus producing anatoxins and Nodularia producing nodularin. Anatoxin concentrations were highly variable and differed significantly among sites; however, this variability did not extend to anatoxin quotas, indicating that the abundance of toxic cells also drives anatoxin content in this system. The anatoxin congener dihydroanatoxin-a comprised a significant proportion of the total anatoxins (38%–71%), indicating that this congener should be included in monitoring. Mats dominated by the green alga Cladophora glomerata contained both anatoxins and nodularin; thus, they may pose an exposure risk to cyanotoxins.  Microcoleus-dominated mats grow in waters with moderate concentrations of dissolved inorganic nitrogen and low concentrations of dissolved reactive phosphorus. Acquisition of phosphorus from organic sources, in addition to within-mat nutrient cycling, may explain how Microcoleus can obtain high biomass in low nutrient environments. In chapter 4, I compare the alkaline phosphatase activity of four toxic and four non-toxic strains under four phosphorus regimes to identify the potential for Microcoleus to use organic phosphorus as a nutrient source. Toxic strains exhibited greater alkaline phosphatase activity than non-toxic strains; however, considerable variability was evident among strains with the same toxigenicity. Environmental mat samples were collected from sites on an upstream-downstream gradient and across a diel cycle, and alkaline phosphatase activity measured. There was a significant difference in alkaline phosphatase activity between the most upstream and downstream site. No diel changes in alkaline phosphatase activity were apparent. The presence of alkaline phosphatase activity suggests that organic phosphorus sources may be accessible to Microcoleus.   To date, management of, and research on, toxic, benthic cyanobacterial proliferations has been focused largely on identifying drivers of growth and toxin production. As a result, there is a significant lack of information on the effects of Microcoleus-dominated proliferations on the wider ecosystem. In chapter 5, an ecotoxicological study was undertaken on Deleatidium spp. larvae using purified anatoxins to address this knowledge gap. Deleatidium spp. larvae were exposed to a range of doses of anatoxin-a, dihydroanatoxin-a, or a mixture of homoanatoxin-a and dihydrohomoanatoxin-a. No significant mortality was observed for any dose or toxin congener. Larvae exposed to high doses (300 μg L⁻¹ to 600 μg L⁻¹) of dihydroanatoxin-a had measurable concentrations of the toxin in their tissues 24-hours post-exposure. The lack of mortality observed, combined with detectable anatoxins in tissues post-exposure, is indicative of a potential pathway for anatoxin transfer or bioaccumulation, and warrants further investigation.  Overall, this thesis has addressed a number of critical knowledge gaps in our understanding of Microcoleus. Continued effort is needed to broaden our understanding of the physiological and ecological role of anatoxins to improve risk assessments and ultimately inform better management of Microcoleus proliferations.</p>