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

We have used serial filtration to isolate picocyanobacteria from brackish and marine microhabitats for analysis. We used 16s metabarcoding to confirm the picocyanobacteria as members of the Order Synechococcales, Genus Cyanobium 6307 (Upper Chilmark Pond) and differing abundances of Cyanobium 6307 and Synechococcus 9902 (Chilmark Pond, Edgartown Great Pond, Tisbury Great Pond and Tashmoo Pond). The proportion and composition of (pico)cyanobacteria in water samples were influenced by the salinity concentrations at various sites, as evidenced by fluorometry and 16s metabarcoding analysis. The cyanobacterial neurotoxin anatoxin-a was present in the picocyanobacterial samples from all studied sites. Additional analyses using fluorometry and 16s metabarcoding described members of the Order Nostocales, including a halotolerant population of Dolichospermum sp., Sphaerospermopsis spp. and Nodularia spp. in Upper Chilmark Pond. We were able to establish a positive linear correlation between cyanobacterial biomass (phycocyanin) and anatoxin-a concentrations using samples taken from Upper Chilmark Pond.

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

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.

Alternative Methods for Analysis of Cyanobacterial Populations in Drinking Water Supplies: Fluorometric and Toxicological Applications Using Phycocyanin

The management of cyanobacteria and potential exposure to associated biotoxins requires the allocation of scarce resources across a range of freshwater resources within various jurisdictions. Cost effective and reliable methods for sample processing and analysis form the foundation of the protocol yielding reliable data from which to derive important decisions. In this study the utilization of new methods to collect, process and analyze samples enhanced our ability to evaluate cyanobacterial populations. Extraction of phycocyanin using the single freeze thaw method provided more accurate and precise measurements (CV 4.7% and 6.4%), offering a simple and cost-effective means to overcome the influence of morphological variability. In-vacuo concentration of samples prior to ELISA analysis provided a detection limit of 0.001 μg·L?1 MC. Fractionation of samples (?1) = ?0.279 + (1.368 ? Log PC (μg·L?1) while in an Aphanizomemon spp. dominant system Log MC (ng·L?1) = 0.385 + (0.449 ? Log PC (μg·L?1). These methods and sampling protocol could be used in other aquatic systems across a broader regional landscape to estimate the levels of microcystins.

Evaluation of Size Structure in Freshwater Cyanobacterial Populations: Methods to Quantify Risk Associated with Changes in Biomass and Microcystin Concentrations

Cyanobacterial populations in surface waters, including drinking water supplies and recreational waters, represent an ever present challenge for resource managers. As communities continuously respond to external and internal processes, dynamic profiles of composition, dominance, growth and toxigenicity emerge. In this study measures of size structure and biomass, quantified using light microscopy and fluorometry, were used to estimate microcystin concentrations through linear regression analysis. Toxigenic profiles using cyanobacterial biomass were developed for lakes dominated by Microcystis spp. and Dolichospermum spp., influenced by both genus-specific pigment concentrations as well as microcystin concentrations. Community composition (Log %MIC) and biomass were used to describe microcystin concentrations in mixed assemblages, where composition was the first input variable. The accessory photopigment phycocyanin was used to describe the linear relationship between the daily growth and net microcystin production rates in the bloom-forming Microcystis spp. samples, suggesting that this size-fractionated sample may provide indications of potential toxigenicity in the whole lake water sample. Future investigations using fluorometric evaluation of cyanobacterial populations could provide additional applications and metrics for use by resource managers to quantify risk association with elevated cyanotoxin concentrations.

Trophic Interactions between Anadromous Juvenile Alewife (<i>Alosa pseudoharengus</i>) and Cyanobacterial Populations in a Shallow Mesotrophic Pond

Alosa pseudoharengus is an anadromous fish that migrates from marine to freshwaters to spawn. The early larval and juvenile forms are known to be planktivorous, where heavy feeding upon their preferred food source of large crustacean zooplankton often results in changes to composition and size structure within this trophic guild which in turn can result in shifts within the trophic spectrum and a classic trophic cascade. In this study of Lower Mill Pond, Brewster MA, we evaluated the feeding strategy of juvenile Alosa to determine whether juvenile alewife switches to feeding largely on cyanobacteria and whether cyanotoxins microcystin (MC) and β-methlyamino-L-alanine (BMAA) bioaccumulate in their muscle tissue. Within 15 - 30 days of their estimated spawning date, overexploitation of crustacean zooplankton resulted in a shift from planktivory to benthic detritivory for the majority of their life history, although this did not reduce their condition based on weight-length relationships (Log Wwt. = -5.503 + (3.101 × Log Length). Mean MC (0.003 μg·g-1 dwt) and BMAA (4.49 μg·g-1 dwt) concentrations in the muscle tissue of out-migrating juveniles were presumably derived from benthic subsidies, exporting freshwater cyanotoxins and creating a potential transfer to consumer of 0.0012 μg MC and 1.85 μg BMAA. Biodilution of MC and biomagnification of BMAA were observed. Depletion of the crustacean biomass by >95% resulted in an increase in the rotifer biomass, where Log crustacean (μg·L-1 dwt) = - 5.642 - (7.976 × Log rotifer (μg·L-1 dwt), and an increase in the amount of potentially edible <50 μm cyanobacterial biomass (r(8) = - 0.676, p = 0.046). A secondary cascade appears to have been maintained via invertebrate planktivory by Chaoborus spp.; however for a period of time edible cyanobacteria growth exceeded grazing pressure, resulting in a bloom of edible cyanobacteria. Continued grazing resulted in a shift to larger, inedible cyanobacterial communities where late season (October) surface accumulations were observed. The mass occurrence of juvenile Alosa pseudoharengus appears to be coupled to the sequential increases of cyanobacterial biomass via its influence on the trophic spectrum. Overall, the rotifer biomass (μg·L-1) was positively correlated with MC (pg·mL-1) (r(8) = 0.577, p = 0.104), and negatively correlated with BMAA (μg·L-1) (r(8) = - 0.388, p = 0.373) in the edible cyanobacterial fraction of the water column, although neither of these were significant.