Cyanobacterial blooms can modify the dynamic of aquatic ecosystems and have harmful consequences for human activities. Moreover, cyanobacteria can produce a variety of cyanotoxins, including microcystins, but little is known about the role of environmental factors on the prevalence of microcystin producers in the cyanobacterial bloom dynamics. This study aimed to better understand the success of Planktothrix in various environments by unveiling the variety of strategies governing cell responses to sudden changes in light intensity and temperature. The cellular responses (photosynthesis, photoprotection, heat shock response and metabolites synthesis) of four Planktothrix strains to high-light or high-temperature were studied, focusing on how distinct ecotypes (red- or green-pigmented) and microcystin production capability affect cyanobacteria's ability to cope with such abiotic stimuli. Our results showed that high-light and high-temperature impact different cellular processes and that Planktothrix responses are heterogeneous, specific to each strain and thus, to genotype. The ability of cyanobacteria to cope with sudden increase in light intensity and temperature was not related to red- or greenpigmented ecotype or microcystin production capability. According to our results, microcystin producers do not cope better to high-light or high-temperature and microcystin content does not increase in response to such stresses.

The "cyanobacteria and microalgae" collection of the Muséum national d'Histoire naturelle includes, respectively, 870 and 890 living strains maintained in the laboratory. This collection supports fundamental research, particularly in taxonomy and ecophysiology, but also numerous activities for the development of bioactive molecules and expertise in environmental diagnosis. These different activities benefit from the emergence of high-throughput "-omics" approaches, which now offer new possibilities to enable such a collection to respond to the current challenges of both fundamental and targeted research, and of the conservation of biological resources. The conservation of organisms in living collections allows us to explore their biological properties. Among these, the production of bioactive metabolites is a rapidly developing theme. The "cyanobacteria and microalgae" collection is a reference collection for environmental diagnosis.

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

En France, de nombreuses toxines non reglementees sont suivies dans le cadre du reseau de veille d’emergence des biotoxines marines dans les coquillages (EMERGTOX) dont le comite de pilotage est preside par la DGAL et inclut la DGS, la Direction des peches maritimes et de l'aquaculture (DPMA), Sante publique France, l’Anses, l’Ifremer et la Direction de l’eau et de la biodiversite (DEB). Les brevetoxines sont incluses dans ce reseau depuis janvier 2018. Elles ont ete mises en evidence pour la premiere fois en France en novembre 2018 dans des moules en Corse (117 μg/kg de glande digestive). Le prelevement d’eau associe pour le suivi du phytoplancton a montre la presence de Karenia spp. (genre de microalgues dont certaines especes produisent des brevetoxines). Il n’existe pas de limite maximale pour ces toxines dans le reglement (CE) n°853/2004. Les USA, l’Australie/Nouvelle-Zelande et le Mexique appliquent un seuil a 800 μg d'equivalent BTX-2/kg de chair de coquillages. Dans la norme CODEXSTAN 292–2008 (rev.2015) du Codex alimentarius, la limite maximale pour les brevetoxines est de 200 unites souris ou equivalent par kg de chair de mollusque vivant. Dans ce contexte, il a ete demande a l’Anses de fournir des elements de reponse aux questions suivantes : Question 1. Quelles sont les donnees toxicologiques disponibles pour les brevetoxines ? Permettent-elles a l’Anses de proposer des valeurs toxicologiques de reference aigue et chronique par voie orale ? Question 2. Sur la base des donnees toxicologiques identifiees par l’Anses, est-il possible de proposer une valeur guide dans les coquillages au-dela de laquelle des investigations complementaires seraient demandees dans le cadre du reseau EMERGTOX ? Question 3. Quelles modalites de suivi des brevetoxines dans le milieu marin pourraient etre recommandees dans le cadre du reseau EMERGTOX ? Quelles investigations complementaires seraient a realiser en cas de depassement de la valeur guide ? Question 4. Compte tenu des donnees toxicologiques et au vu du contexte, existe-t-il une preoccupation de sante publique aux niveaux de contamination identifies dans certaines zones marines francaises liee a la consommation de coquillages ? Question 5. Existe-t-il une preoccupation sanitaire liee a l’exposition aux brevetoxines par un contact direct avec de l’eau contaminee notamment en cas de baignade ou de pratique d’activites nautiques ou en cas d’exposition par inhalation a des embruns ?

Riche de près d’un siècle d’histoire et de plus de 1600 souches vivantes, la collection de cyanobactéries et microalgues du Muséum national d’Histoire naturelle bénéficie de l’émergence des approches « -omics ». Elle peut ainsi répondre aux enjeux actuels de la recherche, notamment en taxonomie et écophysiologie, et constituer une ressource pour le diagnostic environnemental et l’identification de molécules bioactives.

Recent reports tend to predict the increase of harmful cyanobacteria in water systems worldwide due to the climatic and environmental changes, which would compromise water quality and public health. Among abiotic changes, the higher salinities are expected to promote the growth of some harmful species such as Planktothrix agardhii, which is known to build up blooms in brackish areas. Since P. agardhii is a common cyanotoxin producer (microcystin-producing), we investigated here the growth and tolerance of this species when exposed in vitro to a range of salinity levels, while assessing its microcystins variation and production in batch cultures during a time-frame experiment of 18 days. The study revealed a salt acclimation of the brackish P. agardhii that still produced microcystins in salty cultures while maintaining its growth ability in low to medium salinities (ranged from 0 to 7.5 g L−1). For higher salinity concentrations (10 to 12.5 g L−1), microcystins were still detected, while significantly lower growth rates were obtained during the exponential growth phase. This suggests that moderate to high salt ranges do not inhibit the microcystins production of P. agardhii at least for several weeks. Finally, the predicted remediation perspectives in a context of environment salinization assumed by environmental policies may be insufficient to eradicate this potential toxic cyanobacteria, especially when this species is already dominant in the waterbodies.

Efficient RNA extraction methods are needed to study transcript regulation. Such methods must lyse the cell without degrading the genetic material. For cyanobacteria this can be particularly challenging because of the presence of the cyanobacteria cell envelope. The great breath of cyanobacterial shape and size (unicellular, colonial, or filamentous multicellular) created a variety of cell lysis methods. However, there is still a lack of reliable techniques for nucleic acid extraction for several types of cyanobacteria. Here we designed and tested 15 extraction methods using physical, thermic or chemical stress on the filamentous cyanobacteria Planktothrix agardhii. Techniques based on the use of beads, sonication, and heat shock appeared to be too soft to break the Planktothrix agardhii cell envelope, whereas techniques based on the use of detergents degraded the cell envelope but also the RNA. Two protocols allowed to successfully obtain good-quality RNA. The first protocol consisted to manually crush the frozen cell pellet with a pestle and the second was based on the use of high-intensity ultra-sonication. When comparing these two, the high-intensity ultra-sonication protocol was less laborious, faster and allowed to extract 3.5 times more RNA compared to the liquid nitrogen pestle protocol. The high-intensity ultra-sonication protocol was then tested on five Planktothrix strains, this protocol allowed to obtain >8.5 μg of RNA for approximatively 3.5 × 108 cells. The extracted RNA were characterized by 260/280 and 260/230 ratio > to 2, indicating that the samples were devoid of contaminant, and RNA Quality Number > to 7, meaning that the integrity of RNA was preserved with this extraction method. In conclusion, the method we developed based on high-intensity ultra-sonication proved its efficacy in the extraction of Planktothrix RNA and could be helpful for other types of samples.

Recent reports tend to predict the increase of harmful cyanobacteria in water systems worldwide due to the climatic and environmental changes, which would compromise water quality and public health. Among abiotic changes, the higher salinities are expected to promote the growth of some harmful species such asPlanktothrix agardhii, which is known to build up blooms in brackish areas. SinceP. agardhiiis a common cyanotoxin producer (microcystin-producing), we investigated here the growth and tolerance of this species when exposedin vitroto a range of salinity levels, while assessing its microcystins variation and production in batch cultures during a time-frame experiment of 18 days. The study revealed a salt acclimation of the brackishP. agardhiithat still produced microcystins in salty cultures while maintaining its growth ability in low to medium salinities (ranged from 0 to 7.5 g L−1). For higher salinity concentrations (10 to 12.5 g L−1), microcystins were still detected, while significantly lower growth rates were obtained during the exponential growth phase. This suggests that moderate to high salt ranges do not inhibit the microcystins production ofP. agardhiiat least for several weeks. Finally, the predicted remediation perspectives in a context of environment salinization assumed by environmental policies may be insufficient to eradicate this potential toxic cyanobacteria, especially when this species is already dominant in the waterbodies.

Proliferations of cyanobacteria are becoming increasingly widespread in many artificial reservoirs, and may have detrimental effects on ecosystem functioning, especially when the water sustains commercial fisheries. This is the case of the Bir M'Cherga reservoir in North Tunisia, where sporadic fish die-offs have been recently reported. A two-year survey investigated a dual-community structure including both phytoplankton and the main fecal bacterial indicators facing to environmental factors.Low abundances of fecal indicator bacteria were recorded, indicating that no direct human contamination had occurred. However, the ecological status of this reservoir did show signs of degradation, Bacillariophyceae being superseded by Cyanobacteria, with a "nearly-exclusive" dominance of Planktothrix agardhii lasting several months, in association with Planktolyngbya limnetica and Pseudanabaena limnetica. In contrast, the rapid decline of P. agardhii observed in spring 2007 favored greater phytoplankton diversity, with the summer occurrence of Cylindrospermopsis raciborskii. Even though no significant relationships were identified between fish mortality and biological factors, the very presence of two bloom-forming and potentially toxic cyanobacterial species may be viewed as a potentially serious issue with regard to water use and fish farming in the Tunisian reservoir.

Planktothrix agardhii is one of the freshwater cyanobacteria that can produce the hepatotoxin microcystins (MC)—a real threat to human and animal health. Knowledge of the biological role of MC in producing organisms is highly desired to understand the driving force of MC production. Recently, emerging evidences have suggested that MC may have protective role in cells facing environmental stress. If this is true, one should expect differences in the cellular protective mechanisms between MC-containing and MC-deficient mutant strains. To test this hypothesis, it would be essential to investigate the consequences of the loss of MC in Planktothrix in the transcriptional responses of its heat shock proteins (Hsps) to abiotic stresses—an important component of cellular stress response. However, a crucial first step is prerequisite for the isolation of hsp genes here, as the genome of Planktothrix has not been fully published. Therefore, we have successfully isolated four hsp genes including clpC (hsp100), htpG (hsp90), groEL (hsp60), and groES (hsp10) from Planktothrix agardhii PCC 7805 using ramped annealing PCR (RAN-PCR) with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) and annealing control primer (ACP) system. In addition, some putative regulatory sequences found in the upstream region of groESL operon of Planktothrix agardhii were also discussed.

Proliferations of cyanobacteria have detrimental effects on ecosystem functioning, and on the global freshwater food chain. Many studies have focused on the "in situ" dynamics of bloom-forming cyanobacteria, including Cylindrospermopsis raciborskii and Planktothrix agardhii. Few have used experimental assays to explore the fast-growing ability of naturally co-occurring species. Here we investigated the growth of these species when exposed separately (i.e., in monocultures) to a range of light and nutrient conditions, plus their interactive performances in mixed cultures in a short-time experiment (6 days). The use of microplates made it possible to carry out multiple measurements of in-vivo fluorescence (IVF), and to monitor species-dependent biovolumes. No allelopathic effect was significantly observed for any target species, while significantly lower growth rates were obtained in mixed cultures, which may reflect other interference interactions between the species. We showed that Planktothrix grew faster with low light intensity and high nutrient concentrations, and was drastically inhibited by nitrogen deprivation, in contrast to Cylindrospermopsis. However, Cylindrospermopsis outgrew Planktothrix at high NH4+ concentrations, suggesting that this species may be a serious competitor for the native species in many water systems.

Microcystins (MCs) are the most commonly-reported hepatotoxins produced by various cyanobacterial taxa in fresh waters to constitute a potential threat to human and animal health. The biological role of MCs in the producer organisms is not known, and it would be very useful to understand the driving force behind the toxin production. Recent studies have suggested that MCs may have a protective function in cells facing environmental stress. Following this starting premise, we speculate that under adverse conditions the expression of stress-related genes coding for Heat Shock Proteins (Hsp) might be different in an MC-producing strain and its MC-deficient mutant. We therefore used RT-qPCR to compare the expression of 13 hsp genes of an MC-producing strain of Planktothrix agardhii (CYA126/8) and its MC-deficient ΔmcyD mutant over different periods of exposure to high light stress (HL). Three reference genes (RGs) were selected from six candidates to normalize the RT-qPCR data. Of these three RGs (rsh, rpoD, and gltA), gltA is used here for the first time as an RG in prokaryotes. Under HL stress, five genes were found to be strongly up-regulated in both strains (htpG, dnaK, hspA, groES, and groEL). Unexpectedly, we found that the MC-producing wild type strain accumulated higher levels of htpG and dnaK transcripts in response to HL stress than the MC-deficient mutant. In addition, a significant increase in the mcyE transcript was detected in the mutant, suggesting that MCs are required under HL conditions. We discuss several possible roles of MCs in the response to HL stress through their possible involvement in the protective mechanisms of the cells.

We examined the phylogenetic assignment of the Chinese strain PCC 9901, a cyanobacterium previously identified as Lyngbya laxespiralis, using phenotypic and molecular approaches. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain PCC 9901 clustered with Arthrospira rather than Lyngbya. While all the Arthrospira strains were assigned to four genotypes (clusters IA, IB, IIA and IIB) based on the 16S-23S internal transcribed spacer (ITS) rRNA sequences, we reported here an unexpected new genotype for strain PCC 9901 that we named cluster III. A mosaic-like structure was found in two of the three intergenic spacer regions of the ITS (ISR1 and ISR3). In addition, a short repeated sequence was specific for genotype I, and a motif was found in genotype II sequences, but they were absent from the PCC 9901 ITS sequence, suggesting multiple recombination events and/or divergent evolution. Strain PCC 9901 was therefore placed into Arthrospira, and it constituted a new ITS cluster, together with other Asian strains.

The proliferation of microcystins (MCs)-producing cyanobacteria (MCs) can have detrimental effects on the food chain in aquatic environments. Until recently, few studies had focused on the fate of MCs in exposed organisms, such as primary consumers of cyanobacteria. In this study, we investigate the impact of an MC-producing strain of the cyanobacterium Planktothrix agardhii on the growth and physiology of a Nassula sp. ciliate isolated from a non-toxic cyanobacterial bloom. We show that this Nassula sp. strain was able to consume and grow while feeding exclusively on an MC-producing cyanobacterium over a prolonged period of time (8 months). In short-term exposure experiments (8 days), ciliates consuming an MC-producing cyanobacterial strain displayed slower growth rate and higher levels of antioxidant enzymes than ciliates feeding on two non-MC-producing strains. Three high-performance methods (LC/MS, LC/MS-MS and ELISA) were used to quantify the free and bound MCs in the culture medium and in the cells. We show that ciliate grazing led to a marked decrease in free MCs (methanol extractable) in cells, the MCs were therefore no longer found in the surrounding culture medium. These findings suggest that MCs may have undergone redistribution (free vs bound MCs) or chemical degradation within the ciliates.

As previously demonstrated the medaka fish appears to offer a good model for studies of microcystins (MCs) effects. Since cyanobacterial toxins are released with other molecules in the aquatic environment when the producers are dying, in this study, we performed additional experiments in order to compare the described effects obtained with the pure toxin microcystin-LR (MC-LR), among the most toxic MCs, to those induced by complex extracts of an MCs-producer Planktothrix agardhii, strain PMC 75.02 and a natural bloom containing the MCs-producer P. agardhii. The toxicity of these extracts containing several variants of MC was determined in adult medaka treated by gavage. Extracts of an MCs-free strain of P. agardhii (PMC 87.02) were assayed for comparison. Extracts effects were analysed on two tissues, liver and intestine by means of photon and transmission electron microscopy. MC was localized in these tissues by immunocytochemistry. No effect was detectable with extracts of the MCs-free P. agardhii strain. The two MCs-P. agardhii extracts (strain and natural bloom) were able to induce harmful effects in the liver and intestine of the medaka fish in acute intoxication by gavage. In these target organs as shown by toxin immunolocalization, reactions leading to cell disjunction and lysis were observed apparently associated with an immune reaction implying MC containing macrophages. These effects are similar to those previously described with photonic microscopy in medaka treated with pure MC-LR with additional results obtained under the electron microscope. Since no significant effect was detected with the MCs-free (PMC 87.02) extract, we then conclude that MCs, even in complex association with other cyanobacterial components, should be responsible for the toxic effects observed in treated fish.

Selected strains of filamentous Phormidium-like cyanobacteria isolated from two Arctic regions (Ellesmere Island, High Canadian Arctic and Svalbard) and from Antarctica (Antarctic peninsula, South Shetland Islands and South Orkney Islands) were studied. The polyphasic approach used included phenotypic observations of morphological features and genotypic analyses (restriction fragment length polymorphism of 16S rRNA gene, internal transcribed space, 16S rRNA gene sequence analysis). Although genotypes generally correspond to observed morphotypes, the genetic analyses revealed a high degree of biodiversity that could not be unveiled using solely morphological evaluations. According to the phylogenetic analysis, the three clones were divided into two major clades, indicating that the phylogenetic distance between Arct-Ph5/Ant-Ph68 and Ant-Ph58 was so large they belonged to different genera. The polyphyletic position of strains of the genus Phormidium was confirmed by this study, attesting the need to entirely revise classification in this taxon in the future.

Uptake of several naturally occurring organic solutes by the unicellular cyanobacterium Microcystis sp. caused changes in cell turgor pressure (pt), which was determined by measuring the mean critical pressure (pc) of gas vesicles in the cells. Cells had an initial pt of 0.34 MPa, which decreased to 0.08 MPa in 0.15 M sucrose. In solutions of polyols, pt gradually recovered as the solutes penetrated the cytoplasmic membranes. From measurements of the exponential rate of turgor increase, cell volume and surface area, the permeability coefficient of the cytoplasmic membrane to each solute was calculated. Permeabilities to amino acids, ammonium ions and sodium acetate indicated little passive movement of these substances across the cell surface from solutions at high concentrations. We looked for evidence of ion trapping of acetic acid: at low pH there was a rapid rise in turgor pressure indicating a rapid uptake of this weak acid. After 20 min the turgor was lost, apparently due to loss of integrity of the cell membranes. For cells in natural habitats, studies of the permeability of cells to solutes is relevant to the problem of retaining substances that are accumulated by active uptake from solutions of low concentrations in natural waters.

Spatial distribution of the firmly attached epiphytic algae was investigated directly on the leaves of one Apiaceae (Apium nodiflorum L.) commonly found in chalky Mediterranean rivers, at two sampling dates (summer and autumn). The composition and structure of the epiphytic communities of the samples were determined at different levels: along the leaves, on both their sides and within each leaflet (n = 13). Forty-eight leaflets from four randomly selected plants were cut along the longitudinal and vertical axes so as to define 16 microhabitats located on the edge, the centre and the vein. Only a few species of algae were found to be constantly present whatever the sampling date, and dominated all microzones (n = 768). Their presence seemed to depend, rather, on the architectural structure of the plant. The other algae represented the major part of the epiphytic community and were only occasionally present. Those were more subject to temporal and environmental variations and less specific of the substrate. The epiphytes showed considerable spatial heterogeneity within their microhabitats, with an evident preference for the centre and the edge of the leaves, whereas two inhospitable spaces were the vein and apex. On the other hand, when the study was performed at a larger scale (along the leaf), the epiphytic populations of the youngest leaves (near the surface) and the eldest ones (close to the sediments) were homogenous, hence suggesting that other factors, independent from the physiology of the plant, may have an impact on epiphytic distribution.