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We aimed to evaluate the ability of naturally occurring colonies of Microcystis, embedded in a thick mucilage, to persist in estuarine waters. In two batch experiments, we examined the dynamics of microbial communities, including cyanobacteria and associated heterotrophic bacteria, sampled from the field during both a cyanobacterial bloom (non-limiting nutrient condition) and the post-bloom period (limiting nutrient condition), and subjected them to a salinity gradient representative of the freshwater-marine continuum. We demonstrated that both Microcystis aeruginosa and M. wesenbergii survived high salinities due to osmolyte accumulation. Specifically, prolonged exposure to high salinity led to betaine accumulation in the cyanobacterial biomass. The relative abundance of the mcyB gene remained around 30%, suggesting no selection for toxic genotypes with salinity or nutrient changes. Microcystins were predominantly intracellular, except at high salinity levels (>15), where more than 50% of the total microcystin concentration was extracellular. In both nutrient conditions, over 70% of the heterotrophic bacterial community belonged to the Gammaproteobacteria family, followed by the Bacteroidota. Bacterial community composition differed in both size fractions, as well as along the salinity gradient over time. Finally, genus-specific core microbiomes were identified and conserved even under highly stressful conditions, suggesting interactions that support community stability and resilience., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Cyanobacteria are a potential threat to aquatic ecosystems and human health because of their ability to produce cyanotoxins, such as microcystins (MCs). MCs are regularly monitored in fresh waters, but rarely in estuarine and marine waters despite the possibility of their downstream export. Over a period of two years, we monthly analyzed intracellular (in phytoplankton) and extracellular (dissolved in water) MCs at five stations along a river continuum from a freshwater reservoir with ongoing cyanobacterial blooms to the coast of Brittany, France. MCs were quantified using two integrative samplers placed at each site: solid phase adsorption toxin tracking (SPATT) samplers for collecting extracellular MCs and caged mussels (Anodonta anatina and Mytilus edulis) filter-feeding on MC-producing cyanobacteria. The MC transfer was demonstrated each year during five months at estuarine sites and sporadically at the marine outlet. SPATT samplers integrated extracellular MCs, notably at low environmental concentrations (0.2 µg/L) and with the same variant profile as in water. The mussel A. anatina highlighted the presence of MCs including at intracellular concentrations below 1 µg/L. M. edulis more efficiently revealed the MC transfer at estuarine sites than water samplings. Bivalves showed the same MC variant profile as phytoplankton samples, but with differential accumulation capacities between the variants and the two species. Using SPATT or bivalves can give a more accurate assessment of the contamination level of a freshwater-marine continuum, in which the MC transfer can be episodic. MC content in M. edulis represents a potent threat to human health if considering updated French guideline values, and particularly the total (free and protein-bound) MC content, highlighting the necessity to include cyanotoxins in the monitoring of seafood originating from estuarine areas., (Copyright © 2021. Published by Elsevier Ltd.)

• M. aeruginosa and M. wesenbergii survived salinities up to 20. • An increase in compatible solutes, particularly trehalose and betaine, occurred along the freshwater-marine continuum. • Salinity played a key role in structuring the heterotrophic bacteria community. • The mucilage-associated microbiome was conserved along the continuum. Due to climate changes and eutrophication, blooms of predominantly toxic freshwater cyanobacteria are intensifying and are likely to colonize estuaries, thus impacting benthic organisms and shellfish farming representing a major ecological, health and economic risk. In the natural environment, Microcystis form large mucilaginous colonies that influence the development of both cyanobacterial and embedded bacterial communities. However, little is known about the fate of natural colonies of Microcystis by salinity increase. In this study, we monitored the fate of a Microcystis dominated bloom and its microbiome along a French freshwater-marine gradient at different phases of a bloom. We demonstrated changes in the cyanobacterial genotypic composition, in the production of specific metabolites (toxins and compatible solutes) and in the heterotrophic bacteria structure in response to the salinity increase. In particular M. aeruginosa and M. wesenbergii survived salinities up to 20. Based on microcystin gene abundance, the cyanobacteria became more toxic during their estuarine transfer but with no selection of specific microcystin variants. An increase in compatible solutes occurred along the continuum with extensive trehalose and betaine accumulations. Salinity structured most the heterotrophic bacteria community, with an increased in the richness and diversity along the continuum. A core microbiome in the mucilage-associated attached fraction was highly abundant suggesting a strong interaction between Microcystis and its microbiome and a likely protecting role of the mucilage against an osmotic shock. These results underline the need to better determine the interactions between the Microcystis colonies and their microbiome as a likely key to their widespread success and adaptation to various environmental conditions. [ABSTRACT FROM AUTHOR]

The presence of microcystins (MCs) is increasingly being reported in coastal areas worldwide. To provide reliable data regarding this emerging concern, reproducible and accurate methods are required to quantify MCs in salt-containing samples. Herein, we characterized methods of extraction and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for nine MCs and one nodularin (NOD) variants in both cyanobacteria (intracellular) and dissolved forms (extracellular). Different approaches have been used to cope with salinity for the extraction of dissolved MCs but none assessed solid phase extraction (SPE) so far. It was found that salt had negligible effect on the SPE recovery of dissolved MCs using the C18 cartridge while an overestimation up to 67% was noted for some variants with a polymeric sorbent. The limits of detection (LOD) and quantification (LOQ) were 1.0–22 and 5.5–124 pg on column for the intracellular toxins, while 0.05–0.81 and 0.13–2.4 ng/mL were obtained for dissolved toxins. Extraction recoveries were excellent for intracellular (89–121%) and good to excellent for extracellular cyanotoxins (73–102%) while matrix effects were considered neglectable (<12% for 16/20 toxin-matrix combinations), except for the two MC-RR variants. The strategy based on the application of a corrective factor to compensate for losses proved useful as the accuracy was satisfactory (73–117% for intra- and 81–139% for extracellular cyanotoxins, bias <10% for 46/60 conditions, with a few exceptions), with acceptable precisions (intra- and inter-days variabilities <11%). We then applied this method on natural colonies of Microcystis spp. subjected to a salt shock, mimicking their estuarine transfer, in order to assess their survival and to quantify their toxins. The colonies of Microcystis spp. had both their growth and photosynthetic activity impaired at salinities from 10, while toxins remained mainly intracellular (>76%) even at salinity 20, suggesting a potential health risk and contamination of estuarine organisms. [Display omitted] • Successful implementation of Solid Phase Extraction for extracellular microcystins and nodularin in (salt)water samples. • Accurate and repeatable methods for analysis of intracellular and extracellular cyanotoxins using a corrective factor. • Application of the analytical methods on natural Microcystis spp. colonies subjected to a salinity shock. • Colonies survived and toxins remained mainly intracellular, highlighting a potential health risk for estuarine organisms. [ABSTRACT FROM AUTHOR]

• This study reports on the transfer of both cyanobacteria and cyanotoxins from a freshwater reservoir to the marine outlet in France. • The very high correlation between the biomass of potentially toxic species and total MC concentrations strongly suggests that the majority of the cyanobacterial present in the estuary were toxic. • Microcystis which dominated the blooms in the freshwater reservoir was the most likely genus responsible for the measured MC concentrations in the estuary, followed by P. agardhii both being relatively resistant to the salinity gradient. • The extracellular contribution to the total MCs increased from upstream to downstream in accordance with cells lysis at elevated salinity. • Both intracellular and extracellular MC variants did not show specific selection along the salinity gradient but the co-dominance of the highly toxic MC-LR and MC-YR variants is worrisome as it could impact on cyanobacterial consumers. The frequency of cyanobacterial proliferations in fresh waters is increasing worldwide and the presence of associated cyanotoxins represent a threat for ecosystems and human health. While the occurrence of microcystin (MC), the most widespread cyanotoxin, is well documented in freshwaters, only few studies have examined its occurrence in estuarine waters. In this study we evaluated the transfer of cyanobacteria and cyanotoxins along a river continuum from a freshwater reservoir through an interconnecting estuary to the coastal area in Brittany, France. We sampled regularly over 2 years at 5 stations along the river continuum and analysed for phytoplankton and cyanotoxins, together with physico-chemical parameters. Results show that cyanobacteria dominated the phytoplanktonic community with high densities (up to 2 × 106 cells mL−1) at the freshwater sites during the summer and autumn periods of both years, with a cell transfer to estuarine (up to 105 cells mL−1) and marine (2 × 103 cells mL−1) sites. While the temporal variation in cyanobacterial densities was mainly associated with temperature, spatial variation was due to salinity while nutrients were non-limiting for cyanobacterial growth. Cyanobacterial biomass was dominated by several species of Microcystis that survived intermediate salinities. Intracellular MCs were detected in all the freshwater samples with concentrations up to 60 μg L−1, and more intermittently with concentrations up to 1.15 μg L−1, at the most upstream estuarine site. Intracellular MC was only sporadically detected and in low concentration at the most downstream estuarine site and at the marine outlet (respectively <0.14 μg L-1 and <0.03 μg L−1). Different MC variants were detected with dominance of MC-LR, RR and YR and that dominance was conserved along the salinity gradient. Extracellular MC contribution to total MC was higher at the downstream sites in accordance with the lysing of the cells at elevated salinities. No nodularin (NOD) was detected in the particulate samples or in the filtrates. [ABSTRACT FROM AUTHOR]