Genetic association of toxin production in the dinoflagellate Alexandrium minutum.

Dinoflagellates of the genus Alexandrium are responsible for harmful algal blooms and produce paralytic shellfish toxins (PSTs). Their very large and complex genomes make it challenging to identify the genes responsible for toxin synthesis. A family-based genomic association study was developed to determine the inheritance of toxin production in Alexandrium minutum and identify genomic regions linked to this production. We show that the ability to produce toxins is inheritable in a Mendelian way, while the heritability of the toxin profile is more complex. We developed the first dinoflagellate genetic linkage map. Using this map, several major results were obtained: 1. A genomic region related to the ability to produce toxins was identified. 2. This region does not contain any polymorphic sxt genes, known to be involved in toxin production in cyanobacteria. 3. The sxt genes, known to be present in a single cluster in cyanobacteria, are scattered on different linkage groups in A. minutum. 4. The expression of two sxt genes not assigned to any linkage group, sxtI and sxtG, may be regulated by the genomic region related to the ability to produce toxins. Our results provide new insights into the organization of toxicity-related genes in A. minutum, suggesting a dissociated genetic mechanism for the production of the different analogues and the ability to produce toxins. However, most of the newly identified genes remain unannotated. This study therefore proposes new candidate genes to be further explored to understand how dinoflagellates synthesize their toxins.

Sediment archives reveal irreversible shifts in plankton communities after World War II and agricultural pollution.

To evaluate the stability and resilience1 of coastal ecosystem communities to perturbations that occurred during the Anthropocene,2 pre-industrial biodiversity baselines inferred from paleoarchives are needed.3,4 The study of ancient DNA (aDNA) from sediments (sedaDNA)5 has provided valuable information about past dynamics of microbial species6-8 and communities9-18 in relation to ecosystem variations. Shifts in planktonic protist communities might significantly affect marine ecosystems through cascading effects,19-21 and therefore the analysis of this compartment is essential for the assessment of ecosystem variations. Here, sediment cores collected from different sites of the Bay of Brest (northeast Atlantic, France) allowed ca. 1,400 years of retrospective analyses of the effects of human pollution on marine protists. Comparison of sedaDNA extractions and metabarcoding analyses with different barcode regions (V4 and V7 18S rDNA) revealed that protist assemblages in ancient sediments are mainly composed of species known to produce resting stages. Heavy-metal pollution traces in sediments were ascribed to the World War II period and coincided with community shifts within dinoflagellates and stramenopiles. After the war and especially from the 1980s to 1990s, protist genera shifts followed chronic contaminations of agricultural origin. Community composition reconstruction over time showed that there was no recovery to a Middle Ages baseline composition. This demonstrates the irreversibility of the observed shifts after the cumulative effect of war and agricultural pollutions. Developing a paleoecological approach, this study highlights how human contaminations irreversibly affect marine microbial compartments, which contributes to the debate on coastal ecosystem preservation and restoration.

Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates.

Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype and the expression of the alkaline phosphatase activity (APA) is a fundamental phytoplankton strategy for maintaining growth under phosphate-limited conditions. Our aim was to compare the APA among cells and species revived from sediments of the Bay of Brest (Brittany, France), corresponding to a pre-eutrophication period (1940's) and a beginning of a post-eutrophication period (1990's) during which phosphate concentrations have undergone substantial variations. Both toxic marine dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Scrippsiella acuminata were revived from ancient sediments. Using microfluidics, we measured the kinetics of APA at the single-cell level. Our results indicate that all S. acuminata strains had significantly higher APA than A. minutum strains. For both species, the APA in the 1990's decade was significantly lower than in the 1940's. For the first time, our results reveal both inter and intraspecific variabilities of dinoflagellate APA and suggest that, at a half-century timescale, two different species of dinoflagellate may have undergone similar adaptative evolution to face environmental changes and acquire ecological advantages.

Revival of Ancient Marine Dinoflagellates Using Molecular Biostimulation.

The biological processes involved in the preservation, viability, and revival of long-term dormant dinoflagellate cysts buried in sediments remain unknown. Based on studies of plant seed physiology, we tested whether the revival of ancient cysts preserved in century-old sediments from the Bay of Brest (France) could be stimulated by melatonin and gibberellic acid, two molecules commonly used in seed priming. Dinoflagellates were revived from sediments dated to approximately 150 years ago (156 ± 27, 32 cm depth), extending the known record age of cyst viability previously established as around one century. A culture suspension of sediments mixed with melatonin and gibberellic acid solutions as biostimulants exhibited germination of 11 dinoflagellate taxa that could not be revived under controlled culture conditions. The biostimulants revived some dinoflagellates from century-old sediments, including the potentially toxic species Alexandrium minutum. The biostimulants showed positive effects on germination on even more ancient cysts, showing dose-dependent effects on the germination of Scrippsiella acuminata. Concentrations of 1, 10, and 100 µM melatonin and gibberellic acid promoted germination. In contrast, 1,000 µM solutions, particularly for melatonin, drastically decreased germination, suggesting a potential noxious effect of high doses of these molecules on dinoflagellate revival. Our findings suggest that melatonin and gibberellic acid are involved in the stimulation of germination of dinoflagellate cysts. These biostimulants can be used to germinate long-term stored dinoflagellate cysts, which may promote studies of ancient strains in the resurrection ecology research field.

Inter- and Intra-Specific Transcriptional and Phenotypic Responses of Pseudo-nitzschia under Different Nutrient Conditions.

Untangling the functional basis of divergence between closely related species is a step toward understanding species dynamics within communities at both the evolutionary and ecological scales. We investigated cellular (i.e., growth, domoic acid production, and nutrient consumption) and molecular (transcriptomic analyses) responses to varying nutrient concentrations across several strains belonging to three species of the toxic diatom genus Pseudo-nitzschia. Three main results were obtained. First, strains from the same species displayed similar transcriptomic, but not necessarily cellular, responses to the experimental conditions. It showed the importance of considering intraspecific diversity to investigate functional divergence between species. Second, a major exception to the first finding was a strain recently isolated from the natural environment and displaying contrasting gene expression patterns related to cell motility and domoic acid production. This result illustrated the profound modifications that may occur when transferring a cell from the natural to the in vitro environment and asks for future studies to better understand the influence of culture duration and life cycle on expression patterns. Third, transcriptomic responses were more similar between the two species displaying similar ecology in situ, irrespective of the genetic distance. This was especially true for molecular responses related to TCA cycle, photosynthesis, and nitrogen metabolism. However, transcripts related to phosphate uptake were variable between species. It highlighted the importance of considering both overall genetic distance and ecological divergence to explain functional divergence between species.

Revision of the Genus Micromonas Manton et Parke (Chlorophyta, Mamiellophyceae), of the Type Species M. pusilla (Butcher) Manton & Parke and of the Species M. commoda van Baren, Bachy and Worden and Description of Two New Species Based on the Genetic and Phenotypic Characterization of Cultured Isolates.

The green picoalgal genus Micromonas is broadly distributed in estuaries, coastal marine habitats and open oceans, from the equator to the poles. Phylogenetic, ecological and genomic analyses of culture strains and natural populations have suggested that this cosmopolitan genus is composed of several cryptic species corresponding to genetic lineages. We performed a detailed analysis of variations in morphology, pigment content, and sequences of the nuclear-encoded small-subunit rRNA gene and the second internal transcribed spacer (ITS2) from strains isolated worldwide. A new morphological feature of the genus, the presence of tip hairs at the extremity of the hair point, was discovered and subtle differences in hair point length were detected between clades. Clear non-homoplasious synapomorphies were identified in the small-subunit rRNA gene and ITS2 spacer sequences of five genetic lineages. These findings lead us to provide emended descriptions of the genus Micromonas, of the type species M. pusilla, and of the recently described species M. commoda, as well as to describe 2 new species, M. bravo and M. polaris. By clarifying the status of the genetic lineages identified within Micromonas, these formal descriptions will facilitate further interpretations of large-scale analyses investigating ecological trends in time and space for this widespread picoplankter.

Inter and intra-specific growth and domoic acid production in relation to nutrient ratios and concentrations in Pseudo-nitzschia: phosphate an important factor.

The factors responsible for inducing the synthesis of toxins and responses from toxic phytoplankton blooms remain unclear. In this study we compare the influence of genotypic (at both the intra and interspecific levels) and environmental factors (nutrient concentration and ratio) on growth (in terms of cell densities) and domoic acid (DA) production in three Pseudo-nitzschia species: P. australis, P.pungens and P.fradulenta. A strong phosphate effect was detected. More precisely, a low initial concentration in phosphate, even at high initial nitrogen and silicate concentrations, induced the highest DA concentrations and the lowest cell densities in all strains/species studied. In contrast, a low initial concentration of nitrogen and silicate combined, with a higher phosphate concentration resulted in low cell densities, but without high DA production. Inter-species effects were also observed in DA production, where P. australis represented the most toxigenic species of all. Intra-specific variations were only moderate, except for a recently isolated P. australis strain, suggesting the influence of time since isolation on the physiology and DA production of Pseudo-nitzschia species. Overall, the lack of strong interaction between environmental and genotypic factors showed that the various genotypes investigated did not extensively diverge in their ability to respond (in terms of DA production and cell densities) to contrasting nutrient supply.