The Abundance of Toxic Genotypes Is a Key Contributor to Anatoxin Variability in Phormidium-Dominated Benthic Mats

Abstract

The prevalence of benthic proliferations of the anatoxin-producing cyanobacterium Phormidium are increasing in cobble-bed rivers worldwide. Studies to date have shown high spatial and temporal variability in anatoxin concentrations among mats. In this study we determined anatoxin quotas (toxins per cell) in field samples and compared these results to the conventionally-used concentrations (assessed per dry weight of mat). Three mats were selected at sites in two rivers and were sampled every 2–3 h for 24–26 h. The samples were lyophilized and ground to a fine homogenous powder. Two aliquots of known weights were analyzed for anatoxin congeners using liquid chromatography-mass spectrometry, or digital droplet PCR with Phormidium-specific anaC primers to measure absolute quantities of gene copies. Anatoxin concentrations in the mats varied 59- and 303-fold in the two rivers over the study periods. A similar pattern was observed among gene copies (53- and 2828-fold). When converted to anatoxin quotas there was markedly less variability (42- and 16-fold), but significantly higher anatoxin quotas were observed in mats from the second river (p < 0.001, Student’s t-test). There were no obvious temporal patterns with high and low anatoxin concentrations or quotas measured at each sampling time and across the study period. These results demonstrate that variability in anatoxin concentrations among mats is primarily due to the abundance of toxic genotypes. No consistent modulation in anatoxin production was observed during the study, although significant differences in anatoxin quotas among rivers suggest that site-specific physiochemical or biological factors may influence anatoxin production.

Multiple cyanotoxin congeners produced by sub-dominant cyanobacterial taxa in riverine cyanobacterial and algal mats

“…All samples were dominated by ATX and dhATX, and not HTX and dhHTX, which contrasts with observations elsewhere: dhATX, dhHTX and HTX typically dominate in New Zealand [8, 15], and in France ATX dominates [29], although dhATX has also been detected [30]. However, Anderson et al [19] used anatoxins in crude extracts of Microcoleus cultures isolated from the Russian river in their toxicological study and dhATX comprised > 99% of the anatoxins, demonstrating that there is likely large strain to strain variability in the variants produced.…”

“…The absolute number of copies of anaC gene were quantified by ddPCR, using the Phor-AnaC primers and probes (S2 Table) according to the methods of Wood and Puddick [15]. Anatoxin quota were calculated as the summed anatoxins per mg dw divided by the anaC copy number per mg dw as described by Kelly et al [20].…”

“…There is uncertainty about which genera in these systems are producing toxins, although Microcoleus has been identified as an anatoxin producer in the Eel River using assembled metagenomes [18], and cultures of Microcoleus isolated from the Russian River also produce anatoxins [19]. Both toxic and non-toxic genotypes occur in the genus Microcoleus , and these genotypes can be present at varying relative abundances in environmental mats [15, 20], resulting in considerable spatial variability in anatoxin concentrations of mats within short distances.…”

Microbial diversity and metabolic potential in cyanotoxin producing cyanobacterial mats throughout a river network

“…Anatoxin-a synthesis starts from proline, a cyclic amino acid, which is transformed by an eight-gene (anaA-H) polyketide synthase (PKS) operon (Méjean et al, 2010(Méjean et al, , 2014Cadel-Six et al, 2009). Not all strains within anatoxin-a producing species contain the anatoxin-a gene operon, and variation between toxin and non-toxin producing strains occurs over small spatial scales ( <1 cm) (Wood et al, 2012;Wood and Puddick, 2017). Concentrations of anatoxin-a can be driven by changes in the abundance of toxinproducing genotypes within a mat, rather than differential toxin production per cell (Wood and Puddick, 2017).…”

“…Not all strains within anatoxin-a producing species contain the anatoxin-a gene operon, and variation between toxin and non-toxin producing strains occurs over small spatial scales ( <1 cm) (Wood et al, 2012;Wood and Puddick, 2017). Concentrations of anatoxin-a can be driven by changes in the abundance of toxinproducing genotypes within a mat, rather than differential toxin production per cell (Wood and Puddick, 2017).…”