Extraction and analysis by liquid chromatography - tandem mass spectrometry of intra- and extracellular microcystins and nodularin to study the fate of cyanobacteria and cyanotoxins across the freshwater-marine continuum.

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.

Metal stresses modify soluble proteomes and toxin profiles in two Mediterranean strains of the distributed dinoflagellate Alexandrium pacificum.

HABs involving Alexandrium pacificum have been reported in metal-contaminated ecosystems, suggesting that this distributed species adapts to and/or can tolerate the effects of metals. Modifications in soluble proteomes and PST contents were characterized in two Mediterranean A. pacificum strains exposed to mono- or polymetallic stresses (zinc, lead, copper, cadmium). These strains were isolated from two anthropized locations: Santa Giusta Lagoon (Italy, SG C10-3) and the Tarragona seaport (Spain, TAR C5-4F). In both strains, metals primarily downregulated key photosynthesis proteins. Metals also upregulated other proteins involved in photosynthesis (PCP in both strains), the oxidative stress response (HSP 60, proteasome and SOD in SG C10-3; HSP 70 in TAR C5-4F), energy metabolism (AdK in TAR C5-4F), neoglucogenesis/glycolysis (GAPDH and PEP synthase in SG C10-3) and protein modification (PP in TAR C5-4F). These proteins, possibly involved in adaptive proteomic responses, may explain the development of these A. pacificum strains in metal-contaminated ecosystems. The two strains showed different proteomic responses to metals, with SG C10-3 upregulating more proteins, particularly PCP. Among the PSTs, regardless of the metal and the strain studied, C2 and GTX4 predominated, followed by GTX5. Under the polymetallic cocktail, (i) total PSTs, C2 and GTX4 reached the highest levels in SG C10-3 only, and (ii) total PSTs, C2, GTX5 and neoSTX were higher in SG C10-3 than in TAR C5-4F, whereas in SG C10-3 under copper stress, total PSTs, GTX5, GTX1 and C1 were higher than in the controls, revealing variability in PST biosynthesis between the two strains. Total PSTs, C2, GTX4 and GTX1 showed significant positive correlations with PCP, indicating that PST production may be positively related to photosynthesis. Our results showed that the A. pacificum strains adapt their proteomic and physiological responses to metals, which may contribute to their ecological success in highly anthropized areas.

Physiological changes induced by sodium chloride stress in Aphanizomenon gracile, Cylindrospermopsis raciborskii and Dolichospermum sp.

Due to anthropogenic activities, associated with climate change, many freshwater ecosystems are expected to experience an increase in salinity. This phenomenon is predicted to favor the development and expansion of freshwater cyanobacteria towards brackish waters due to their transfer along the estuarine freshwater-marine continuum. Since freshwater cyanobacteria are known to produce toxins, this represents a serious threat for animal and human health. Saxitoxins (STXs) are classified among the most powerful cyanotoxins. It becomes thus critical to evaluate the capacity of cyanobacteria producing STXs to face variations in salinity and to better understand the physiological consequences of sodium chloride (NaCl) exposure, in particular on their toxicity. Laboratory experiments were conducted on three filamentous cyanobacteria species isolated from brackish (Dolichospermum sp.) and fresh waters (Aphanizomenon gracile and Cylindrospermopsis raciborskii) to determine how salinity variations affect their growth, photosynthetic activity, pigment composition, production of reactive oxygen species (ROS), synthesis of compatible solutes and STXs intracellular quotas. Salinity tolerance was found to be species-specific. Dolichospermum sp. was more resistant to salinity variations than A. gracile and C. raciborskii. NaCl variations reduced growth in all species. In A. gracile, carotenoids content was dose-dependently reduced by NaCl. By contrast, in C. raciborskii and Dolichospermum sp., variations in carotenoids content did not show obvious relationships with NaCl concentration. While in Dolichospermum sp. phycocyanin and phycoerythrin increased within the first 24 h exposure to NaCl, in both A. gracile and C. raciborskii, these pigments decreased proportionally to NaCl concentration. Low changes in salinity did not impact STXs production in A. gracile and C. raciborskii while higher increase in salinity could modify the toxin profile and content of C. raciborskii (intracellular STX decreased while dc-GTX2 increased). In estuaries, A. gracile and C. raciborskii would not be able to survive beyond the oligohaline area (i.e. salinity > 5). Conversely, in part due to its ability to accumulate compatible solutes, Dolichospermum sp. has the potential to face consequent salinity variations and to survive in the polyhaline area (at least up to salinity = 24).

From the sxtA4 Gene to Saxitoxin Production: What Controls the Variability Among Alexandrium minutum and Alexandrium pacificum Strains?

Paralytic shellfish poisoning (PSP) is a human foodborne syndrome caused by the consumption of shellfish that accumulate paralytic shellfish toxins (PSTs, saxitoxin group). In PST-producing dinoflagellates such as Alexandrium spp., toxin synthesis is encoded in the nuclear genome via a gene cluster (sxt). Toxin production is supposedly associated with the presence of a 4th domain in the sxtA gene (sxtA4), one of the core genes of the PST gene cluster. It is postulated that gene expression in dinoflagellates is partially constitutive, with both transcriptional and post-transcriptional processes potentially co-occurring. Therefore, gene structure and expression mode are two important features to explore in order to fully understand toxin production processes in dinoflagellates. In this study, we determined the intracellular toxin contents of twenty European Alexandrium minutum and Alexandrium pacificum strains that we compared with their genome size and sxtA4 gene copy numbers. We observed a significant correlation between the sxtA4 gene copy number and toxin content, as well as a moderate positive correlation between the sxtA4 gene copy number and genome size. The 18 toxic strains had several sxtA4 gene copies (9-187), whereas only one copy was found in the two observed non-toxin producing strains. Exploration of allelic frequencies and expression of sxtA4 mRNA in 11 A. minutum strains showed both a differential expression and specific allelic forms in the non-toxic strains compared with the toxic ones. Also, the toxic strains exhibited a polymorphic sxtA4 mRNA sequence between strains and between gene copies within strains. Finally, our study supported the hypothesis of a genetic determinism of toxin synthesis (i.e., the existence of several genetic isoforms of the sxtA4 gene and their copy numbers), and was also consistent with the hypothesis that constitutive gene expression and moderation by transcriptional and post-transcriptional regulation mechanisms are the cause of the observed variability in the production of toxins by A. minutum.

Alexandrium pacificum and Alexandrium minutum: Harmful or environmentally friendly?

Alexandrium minutum and Alexandrium pacificum are representatives of the dinoflagellate genus that regularly proliferate on the French coasts and other global coastlines. These harmful species may threaten shellfish harvest and human health due to their ability to synthesize neurotoxic alkaloids of the saxitoxin group. However, some dinoflagellates such as A. minutum, and as reported here A. pacificum as well, may also have a beneficial impact on the environment by producing dimethylsulfoniopropionate-DMSP, the precursor of dimethylsulfur-DMS and sulfate aerosols involved in climate balance. However, environmental conditions might influence Alexandrium physiology towards the production of harmful or environmentally friendly compounds. After assessing the influence of two salinity regimes (33 and 38) relative to each species origin (Atlantic French coast and Mediterranean Lagoon respectively), it appears that DMSP and toxin content was variable between the three experimented strains and that higher salinity disadvantages toxin production and tends to favor the production of the osmolytes DMSP and glycine betaine. Hence, this key metabolite production is strain and species-dependent and is influenced by environmental conditions of salinity which in turn, can diversely affect the environment. Widespread coastal blooms of A. minutum and A. pacificum, although being a risk for seafood contamination with toxins, are also a DMSP and DMS source that potentially contribute to the ecosystem structuration and climate. Regarding recent advances in DMSP biosynthesis pathway, 3 dsyB homologs were found in A. minutum but no homolog of the diatom sequence TpMMT.

Acclimation of the Marine Diatom Pseudo-nitzschia australis to Different Salinity Conditions: Effects on Growth, Photosynthetic Activity, and Domoic Acid Content1.

Toxic Pseudo-nitzschia australis strains isolated from French coastal waters were studied to investigate their capacity to adapt to different salinities. Their acclimation to different salinity conditions (10, 20, 30, 35, and 40) was studied on growth, photosynthetic capacity, cell biovolume, and domoic acid (DA) content. The strains showed an ability to acclimate to a salinity range from 20 to 40, with optimal growth rates between salinities 30 and 40. The highest cell biovolume was observed at the lowest salinity 20 and was associated with the lowest growth rate. Salinity did not affect the photosynthetic activity; Fv /Fm values and the pigment contents remained high with no significant difference among salinities. An enhanced production of zeaxanthin was, however, observed in the late stationary and decline phases in all cultures except for those acclimated to salinity 20. In terms of cellular toxin content, DA concentrations were 2 to 3-fold higher at the lowest salinity (20) than at the other salinities and were combined with a low amount of dissolved DA. The fact that P. australis accumulate more DA per cell in less saline waters, illustrates that climate-related changes in salinity may affect Pseudo-nitzschia physiology through direct effects on growth, physiology, and toxin content.

Influence of sudden salinity variation on the physiology and domoic acid production by two strains of Pseudo-nitzschia australis.

Several coastal countries including France have experienced serious and increasing problems related to Pseudo-nitzschia toxic blooms. These toxic blooms occur in estuarine and coastal waters potentially subject to fluctuations in salinity. In this study, we document for the first time the viability, growth, photosynthetic efficiency, and toxin production of two strains of Pseudo-nitzschia australis grown under conditions with sudden salinity changes. Following salinity variation, the two strains survived over a restricted salinity range of 30-35, with favorable physiological responses, as the growth, effective quantum yield and toxin content were high compared to the other conditions. In addition, high cellular quotas of domoic acid (DA) were observed at a salinity of 40 for the strain IFR-PAU-16.1 in comparison with the other strain IFR-PAU-16.2 where the cell DA content was directly released into the medium. On the other hand, the osmotic stress imposed at lower salinities, 20 and 10, resulted in cell lysis and a sudden DA leakage in the medium. Intra-specific variability was observed in growth and toxin production, with the strain IFR-PAU-16.1 apparently able to withstand higher salinities than the strain IFR-PAU-16.2. On the whole, DA does not appear to act as an osmolyte in response to sudden salinity changes. Since most of the shellfish harvesting areas of bivalve molluscs in France are located in areas where the salinity generally varies between 30 and 35, Pseudo-nitzschia australis blooms might potentially impact public health and commercial shellfish resources in these places.