Isolation of Picocyanobacteria (Order <i>Synechococcales</i>) and Occurrence of the Cyanotoxin Anatoxin-A in a Shallow Mesotrophic Pond

Abstract

We have verified the use of a serial filtration method to isolate picocyanobacteria for analysis. We used eDNA metabarcoding to confirm the picocyanobacteria as members of the Order Synechococcales, Genus Cyanobium, specifically Cyanobium 6307. Fluorometric analysis using accessory pigments phycocyanin and phycoerythrin described periods of excess biomass, where the net growth rate model confirmed these conditions. The total anatoxin-a concentrations in the picocyanobacterial sample ranged from 0.0074 -6.41 µg•L −1 representing a 40-fold difference over the entire sampling season. Sampling frequency of every three days appeared to be an important factor in capturing these changes in anatoxin-a concentration. During a period of excess biomass, we were able to establish a linear correlation between cyanobacterial biomass and Anatoxin-a concentrations.

Isolation of Picocyanobacteria (Order <i>Synechococcales</i>) and Occurrence of Cyanotoxins (Anatoxin-a) in Saline Microhabitats at Martha’s Vineyard, MA

“…Recent genomic [6] and 16s metabarcoding [7] analysis has confirmed that commonly found picocyanobacteria (Order Synechococcales) can produce anatoxin-a. As described elsewhere [7], the presence of (pico)cyanobacteria can be heavily influenced by salinity.…”

“…Samples of whole lake water (WLW) and net (NET) water samples were collected from five different sites representing a range of saline environments on a series of dates between May and October from the shoreline (maximum depth 1m) (Figure 1 brown amber bottles, placed on ice and transported to the laboratory for further analysis. Size fractionation using floatation, serial filtration with gravity and serial filtration with pressure were used for sample processing and collection [7].…”

“…All water samples were prepared, preserved, stored and analyzed as previously described [7]. Prior to fluorometric analysis, the hand-held fluorometry units were calibrated [8] for both phycocyanin (PC) and phycoerythrin (PE), accessory Journal of Water Resource and Protection pigments primarily associated with cyanobacterial (phycocyanin) and picocyanobacterial (phycoerythrin) biomass.…”

“…The whole lake water sample (WLW) was fractionated (<50 µm, <10 µm, <5 µm, <0.2 µm) according to the project QAPP and samples frozen for a single freeze-thaw extraction (SFT) prior to fluorometric analysis using the handheld Fluoroquik TM fluorometer (excitation wavelengths 595 nm for phycocyanin and 545 nm for phycoerythrin) using a quantitation limit for phycocyanin (PC) at 3 µg•L −1 and phycoerythrin (PE) at 0.1 µg•L −1 . The 16s meta-barcoding results were used as a quality assurance/quality control (QA/QC) measure as previously described [7]. The number of reads was used as a measure of (pico)cyanobacterial abundance.…”

“…The picocyanobacteria can produce secondary metabolites including microcystin (MC) and its variants and B-Methylamino-L-alanine (BMAA) [4] [5]. Recent genomic [6] and 16s metabarcoding [7] analysis has confirmed that commonly found picocyanobacteria (Order Synechococcales) can produce anatoxin-a. As described elsewhere [7], the presence of (pico)cyanobacteria can be heavily influenced by salinity.…”