Spatio-temporal connectivity of a toxic cyanobacterial community and its associated microbiome along a freshwater-marine 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.

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.

Production and composition of extracellular polymeric substances by a unicellular strain and natural colonies of Microcystis: Impact of salinity and nutrient stress.

The transfer of toxic cyanobacterial Microcystis blooms from freshwater to estuaries constitutes a serious environmental problem worldwide that is expected to expand in scale and intensity with anthropogenic and climate change. The formation and maintenance of Microcystis in colonial form is conditioned to the presence of extracellular polymeric substances (EPS). In this study, we attempted to better understand how the mucilaginous colonial form of Microcystis evolves under environmental stress conditions. In particular, we studied and compared the production and the composition of EPS fractions (attached and free) from natural colonies of a Microcystis bloom and from a unicellular M. aeruginosa strain under salinity and nutrient stress (representing a land-sea continuum). Our results highlighted a greater production of EPS from the natural colonies of Microcystis than the unicellular one under nutrient and combined stress conditions dominated by the attached form. In comparison to the unicellular Microcystis, EPS produced by the colonial form were characterized by high molecular weight polysaccharides which were enriched in uronic acids and hexosamines, notably for the free fraction in response to increased salinities. This complex extracellular matrix gives the cells the ability to aggregate and allows the colonial cyanobacterial population to cope with osmotic shock.

Morphological and physiological impacts of salinity on colonial strains of the cyanobacteria Microcystis aeruginosa.

In the context of global change and enhanced toxic cyanobacterial blooms, cyanobacterial transfer to estuaries is likely to increase in frequency and intensity and impact animal and human health. Therefore, it is important to evaluate the potential of their survival in estuaries. In particular, we tested if the colonial form generally observed in natural blooms enhanced the resistance to salinity shock compared to the unicellular form generally observed in isolated strains. We tested the impact of salinity on two colonial strains of Microcystis aeruginosa, producing different amounts of mucilage by combining classical batch methods with a novel microplate approach. We demonstrate that the collective organization of these pluricellular colonies improves their ability to cope with osmotic shock when compared to unicellular strains. The effect of a sudden high salinity increase (S ≥ 20) over 5 to 6 days had several impacts on the morphology of M. aeruginosa colonies. For both strains, we observed a gradual increase in colony size and a gradual decrease in intercellular spacing. For one strain, we also observed a decrease in cell diameter with an increase in mucilage extent. The pluricellular colonies formed by both strains could withstand higher salinities than unicellular strains studied previously. In particular, the strain producing more mucilage displayed a sustained autofluorescence even at S = 20, a limit that is higher than the most robust unicellular strain. These results imply survival and possible M. aeruginosa proliferation in mesohaline estuaries.

Deciphering interactions between the marine dinoflagellate Prorocentrum lima and the fungus Aspergillus pseudoglaucus.

The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal-fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal-fungal co-culture metabolomes were monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal-fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal-fungal interactions in the study of parameters regulating toxin production.

Sharing Vitamin B12 between Bacteria and Microalgae Does Not Systematically Occur: Case Study of the Haptophyte Tisochrysis lutea.

Haptophyte microalgae are key contributors to microbial communities in many environments. It has been proposed recently that members of this group would be virtually all dependent on vitamin B12 (cobalamin), an enzymatic cofactor produced only by some bacteria and archaea. Here, we examined the processes of vitamin B12 acquisition by haptophytes. We tested whether co-cultivating the model species Tisochrysis lutea with B12-producing bacteria in vitamin-deprived conditions would allow the microalga to overcome B12 deprivation. While T. lutea can grow by scavenging vitamin B12 from bacterial extracts, co-culture experiments showed that the algae did not receive B12 from its associated bacteria, despite bacteria/algae ratios supposedly being sufficient to allow enough vitamin production. Since other studies reported mutualistic algae-bacteria interactions for cobalamin, these results question the specificity of such associations. Finally, cultivating T. lutea with a complex bacterial consortium in the absence of the vitamin partially rescued its growth, highlighting the importance of microbial interactions and diversity. This work suggests that direct sharing of vitamin B12 is specific to each species pair and that algae in complex natural communities can acquire it indirectly by other mechanisms (e.g., after bacterial lysis).

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).

A community perspective on the concept of marine holobionts: current status, challenges, and future directions.

Host-microbe interactions play crucial roles in marine ecosystems. However, we still have very little understanding of the mechanisms that govern these relationships, the evolutionary processes that shape them, and their ecological consequences. The holobiont concept is a renewed paradigm in biology that can help to describe and understand these complex systems. It posits that a host and its associated microbiota with which it interacts, form a holobiont, and have to be studied together as a coherent biological and functional unit to understand its biology, ecology, and evolution. Here we discuss critical concepts and opportunities in marine holobiont research and identify key challenges in the field. We highlight the potential economic, sociological, and environmental impacts of the holobiont concept in marine biological, evolutionary, and environmental sciences. Given the connectivity and the unexplored biodiversity specific to marine ecosystems, a deeper understanding of such complex systems requires further technological and conceptual advances, e.g., the development of controlled experimental model systems for holobionts from all major lineages and the modeling of (info)chemical-mediated interactions between organisms. Here we propose that one significant challenge is to bridge cross-disciplinary research on tractable model systems in order to address key ecological and evolutionary questions. This first step is crucial to decipher the main drivers of the dynamics and evolution of holobionts and to account for the holobiont concept in applied areas, such as the conservation, management, and exploitation of marine ecosystems and resources, where practical solutions to predict and mitigate the impact of human activities are more important than ever.

Cell free Microcystis aeruginosa spent medium affects Daphnia magna survival and stress response.

Primary consumers in freshwater ecosystems, such as the zooplankton organism Daphnia magna, are highly affected by cyanobacteria, both as they may use it as a food source but also by cyanobacterial metabolites present in the water. Here, we investigate the impacts of cyanobacterial metabolites focussing on the environmental realistic scenario of the naturally released mixture without crushing cyanobacterial cells or their uptake as food. Therefore, D. magna were exposed to two concentrations of cell free cyanobacterial spent medium from Microcystis aeruginosa PCC 7806 to represent higher and lower ecologically-relevant concentrations of cyanobacterial metabolites. Including microcystin-LR, 11 metabolites have been detected of which 5 were quantified. Hypothesising concentration and time dependent negative impact, survival, gene expression marking digestion and metabolism, oxidative stress response, cell cycle and molting as well as activities of detoxification and antioxidant enzymes were followed for 7 days. D. magna suffered from oxidative stress as both catalase and glutathione S-transferase enzyme activities significantly decreased, suggesting enzyme exhaustibility after 3 and 7 days. Moreover, gene-expressions of the 4 stress markers (glutathione S-transferase, glutathione peroxidase, catalase and thioredoxin) were merely downregulated after 7 days of exposure. Energy allocation (expression of glyceraldehyde-3-phosphate dehydrogenase) was increased after 3 days but decreased as well after 7 days exposure. Cell cycle was impacted time dependently but differently by the two concentrations, along with an increasing downregulation of myosin heavy chain responsible for cell arrangement and muscular movements. Deregulation of nuclear hormone receptor genes indicate that D. magna hormonal steering including molting seemed impaired despite no detection of microviridin J in the extracts. As a consequence of all those responses and presumably of more than investigated molecular and physiological changes, D. magna survival was impaired over time, in a concentration dependent manner. Our results confirm that besides microcystin-LR, other secondary metabolites contribute to negative impact on D. magna survival and stress response.

Cross talk: Two way allelopathic interactions between toxic Microcystis and Daphnia.

Due to eutrophication, freshwater ecosystems frequently experience cyanobacterial blooms, many of which produce bioactive metabolites that can affect vertebrates and invertebrates life traits. Zooplankton are able to develop tolerance as a physiological response to cyanobacteria and their bioactive compounds, however, this comes with energetic cost that in turn influence Daphnia life traits and may impair populations. Vice versa, it has been suggested that Daphnia are able to reduce cyanobacterial dominance until a certain cyanobacterial density; it remains unclear whether Daphnia metabolites alone influence the physiological state and bioactive metabolites production of cyanobacteria. Hence, this study investigates mutual physiological reactions of toxic Microcystis aeruginosa PCC7806 and Daphnia magna. We hypothesize that a) the presence of D. magna will negatively affect growth, increase stress response and metabolites production in M. aeruginosa PCC7806 and b) the presence of M. aeruginosa PCC7806 will negatively affect physiological responses and life traits in D. magna. In order to test these hypotheses experiments were conducted in a specially designed co-culture chamber that allows exchange of the metabolites without direct contact. A clear mutual impact was evidenced. Cyanobacterial metabolites reduced survival of D. magna and decreased oxidative stress enzyme activity. Simultaneously, presence of D. magna did not affect photosynthetic activity. However, ROS increase and tendencies in cell density decrease were observed on the same day, suggesting possible energy allocation towards anti-oxidative stress enzymes, or other protection mechanisms against Daphnia infochemicals, as the strain managed to recover. Elevated concentration of intracellular and overall extracellular microcystin MC-LR, as well as intracellular concentrations of aerucyclamide A and D in the presence of Daphnia, indicating a potential protective or anti-grazing function. However, more research is needed to confirm these findings.

Salt Shock Responses of Microcystis Revealed through Physiological, Transcript, and Metabolomic Analyses.

The transfer of Microcystis aeruginosa from freshwater to estuaries has been described worldwide and salinity is reported as the main factor controlling the expansion of M. aeruginosa to coastal environments. Analyzing the expression levels of targeted genes and employing both targeted and non-targeted metabolomic approaches, this study investigated the effect of a sudden salt increase on the physiological and metabolic responses of two toxic M. aeruginosa strains separately isolated from fresh and brackish waters, respectively, PCC 7820 and 7806. Supported by differences in gene expressions and metabolic profiles, salt tolerance was found to be strain specific. An increase in salinity decreased the growth of M. aeruginosa with a lesser impact on the brackish strain. The production of intracellular microcystin variants in response to salt stress correlated well to the growth rate for both strains. Furthermore, the release of microcystins into the surrounding medium only occurred at the highest salinity treatment when cell lysis occurred. This study suggests that the physiological responses of M. aeruginosa involve the accumulation of common metabolites but that the intraspecific salt tolerance is based on the accumulation of specific metabolites. While one of these was determined to be sucrose, many others remain to be identified. Taken together, these results provide evidence that M. aeruginosa is relatively salt tolerant in the mesohaline zone and microcystin (MC) release only occurs when the capacity of the cells to deal with salt increase is exceeded.

Physiological and Metabolic Responses of Freshwater and Brackish-Water Strains of Microcystis aeruginosa Acclimated to a Salinity Gradient: Insight into Salt Tolerance.

Proliferation of microcystin (MC)-producing Microcystis aeruginosa in brackish waters has been described in several locations and represents a new concern for public and environmental health. While the impact of a sudden salinity increase on M. aeruginosa physiology has been studied, less is known about the mechanisms involved in salt tolerance after acclimation. This study aims to compare the physiological responses of two strains of M. aeruginosa (PCC 7820 and PCC 7806), which were isolated from contrasted environments, to increasing salinities. After acclimation, growth and MC production rates were determined and metabolomic analyses were conducted. For both strains, salinity decreased the biovolume, growth, and MC production rates and induced the accumulation of polyunsaturated lipids identified as monogalactosyldiacylglycerol. The distinct salt tolerances (7.5 and 16.9) obtained between the freshwater (PCC 7820) and the brackish-water (PCC 7806) strains suggested different strategies to cope with the osmotic pressure, as revealed by targeted and untargeted metabolomic analyses. An accumulation of trehalose as the main compatible solute was obtained in the freshwater strain, while sucrose was mainly accumulated in the brackish one. Moreover, distinct levels of glycine betaine and proline accumulation were noted. Altogether, metabolomic analysis illustrated a strain-specific response to salt tolerance, involving compatible solute production.IMPORTANCE Blooms of Microcystis aeruginosa and the production of microcystins are major issues in eutrophic freshwater bodies. Recently, an increasing number of proliferations of M. aeruginosa in brackish water has been documented. The occurrence of both M. aeruginosa and microcystins in coastal areas represents a new threat for human and environmental health. In order to better describe the mechanisms involved in Microcystis sp. proliferation in brackish water, this study used two M. aeruginosa strains isolated from fresh and brackish waters. High salinity reduced the growth rate and microcystin production rate of M. aeruginosa In order to cope with higher salinities, the strains accumulated different cyanobacterial compatible solutes, as well as unsaturated lipids, explaining their distinct salt tolerance.

Daphnia magna Exudates Impact Physiological and Metabolic Changes in Microcystis aeruginosa.

While the intracellular function of many toxic and bioactive cyanobacterial metabolites is not yet known, microcystins have been suggested to have a protective role in the cyanobacterial metabolism, giving advantage to toxic over nontoxic strains under stress conditions. The zooplankton grazer Daphnia reduce cyanobacterial dominance until a certain density, which may be supported by Daphnia exudates, affecting the cyanobacterial physiological state and metabolites' production. Therefore, we hypothesized that D. magna spent medium will impact the production of cyanobacterial bioactive metabolites and affect cyanobacterial photosynthetic activity in the nontoxic, but not the toxic strain. Microcystin (MC-LR and des-MC-LR) producing M. aeruginosa PCC7806 and its non-microcystin producing mutant were exposed to spent media of different D. magna densities and culture durations. D. magna spent medium of the highest density (200/L) cultivated for the shortest time (24 h) provoked the strongest effect. D.magna spent medium negatively impacted the photosynthetic activity of M. aeruginosa PCC7806, as well as the dynamics of intracellular and extracellular cyanobacterial metabolites, while its mutant was unaffected. In the presence of Daphnia medium, microcystin does not appear to have a protective role for the strain. On the contrary, extracellular cyanopeptolin A increased in M. aeruginosa PCC7806 although the potential anti-grazing role of this compound would require further studies.

Chemically mediated interactions between Microcystis and Planktothrix: impact on their growth, morphology and metabolic profiles.

Freshwater cyanobacteria are known for their ability to produce bioactive compounds, some of which have been described as allelochemicals. Using a combined approach of co-cultures and analyses of metabolic profiles, we investigated chemically mediated interactions between two cyanobacterial strains, Microcystis aeruginosa PCC 7806 and Planktothrix agardhii PCC 7805. More precisely, we evaluated changes in growth, morphology and metabolite production and release by both interacting species. Co-culture of Microcystis with Planktothrix resulted in a reduction of the growth of Planktothrix together with a decrease of its trichome size and alterations in the morphology of its cells. The production of intracellular compounds by Planktothrix showed a slight decrease between monoculture and co-culture conditions. Concerning Microcystis, the number of intracellular compounds was higher under co-culture condition than under monoculture. Overall, Microcystis produced a lower number of intracellular compounds under monoculture than Planktothrix, and a higher number of intracellular compounds than Planktothrix under co-culture condition. Our investigation did not allow us to identify specifically the compounds causing the observed physiological and morphological changes of Planktothrix cells. However, altogether, these results suggest that co-culture induces specific compounds as a response by Microcystis to the presence of Planktothrix. Further studies should be undertaken for identification of such potential allelochemicals.

Unique Biosynthetic Pathway in Bloom-Forming Cyanobacterial Genus Microcystis Jointly Assembles Cytotoxic Aeruginoguanidines and Microguanidines.

The cyanobacterial genus Microcystis is known to produce an elaborate array of structurally unique and biologically active natural products, including hazardous cyanotoxins. Cytotoxic aeruginoguanidines represent a yet unexplored family of peptides featuring a trisubstituted benzene unit and farnesylated arginine derivatives. In this study, we aimed at assigning these compounds to a biosynthetic gene cluster by utilizing biosynthetic attributes deduced from public genomes of Microcystis and the sporadic distribution of the metabolite in axenic strains of the Pasteur Culture Collection of Cyanobacteria. By integrating genome mining with untargeted metabolomics using liquid chromatography with mass spectrometry, we linked aeruginoguanidine (AGD) to a nonribosomal peptide synthetase gene cluster and coassigned a significantly smaller product to this pathway, microguanidine (MGD), previously only reported from two Microcystis blooms. Further, a new intermediate class of compounds named microguanidine amides was uncovered, thereby further enlarging this compound family. The comparison of structurally divergent AGDs and MGDs reveals an outstanding versatility of this biosynthetic pathway and provides insights into the assembly of the two compound subfamilies. Strikingly, aeruginoguanidines and microguanidines were found to be as widespread as the hepatotoxic microcystins, but the occurrence of both toxin families appeared to be mutually exclusive.

Role of bacteria in the production and degradation of Microcystis cyanopeptides.

The freshwater cyanobacteria, Microcystis sp., commonly form large colonies with bacteria embedded in their mucilage. Positive and negative interactions between Microcystis species and their associated bacteria have been reported. However, the potential role of bacteria in the production and degradation of cyanobacterial secondary metabolites has not been investigated. In this study, a Microcystis-associated bacterial community was isolated and added to the axenic M. aeruginosaPCC7806 liquid culture. After 3 years of cocultivation, we studied the bacterial genetic diversity adapted to the PCC7806 strain and compared the intra- and extracellular concentration of major cyanopeptides produced by the cyanobacterial strain under xenic and axenic conditions. Mass spectrometric analyses showed that the intracellular concentration of peptides was not affected by the presence of bacteria. Interestingly, the produced peptides were detected in the axenic media but could not be found in the xenic media. This investigation revealed that a natural bacterial community, dominated by Alpha-proteobacteria, was able to degrade a wide panel of structurally varying cyclic cyanopeptides.

Changes in secondary metabolic profiles of Microcystis aeruginosa strains in response to intraspecific interactions.

The cyanobacteria Microcystis proliferate in freshwater ecosystems and produce bioactive compounds including the harmful toxins microcystins (MC). These secondary metabolites play an important role in shaping community composition through biotic interactions although their role and mode of regulation are poorly understood. As natural cyanobacterial populations include producing and non-producing strains, we tested if the production of a range of peptides by coexisting cells could be regulated through intraspecific interactions. With an innovative co-culturing chamber together with advanced mass spectrometry (MS) techniques, we monitored the growth and compared the metabolic profiles of a MC-producing as well as two non-MC-producing Microcystis strains under mono- and co-culture conditions. In monocultures, these strains grew comparably; however, the non-MC-producing mutant produced higher concentrations of cyanopeptolins, aerucyclamides and aeruginosins than the wild type. Physiological responses to co-culturing were reflected in a quantitative change in the production of the major peptides. Using a MS/MS-based molecular networking approach, we identified new analogues of known classes of peptides as well as new compounds. This work provides new insights into the factors that regulate the production of MC and other secondary metabolites in cyanobacteria, and suggests interchangeable or complementary functions allowing bloom-forming cyanobacteria to efficiently colonize and dominate in fluctuating aquatic environments.

Evidence of the cost of the production of microcystins by Microcystis aeruginosa under differing light and nitrate environmental conditions.

The cyanobacterium Microcystis aeruginosa is known to proliferate in freshwater ecosystems and to produce microcystins. It is now well established that much of the variability of bloom toxicity is due to differences in the relative proportions of microcystin-producing and non-microcystin-producing cells in cyanobacterial populations. In an attempt to elucidate changes in their relative proportions during cyanobacterial blooms, we compared the fitness of the microcystin-producing M. aeruginosa PCC 7806 strain (WT) to that of its non-microcystin-producing mutant (MT). We investigated the effects of two light intensities and of limiting and non-limiting nitrate concentrations on the growth of these strains in monoculture and co-culture experiments. We also monitored various physiological parameters, and microcystin production by the WT strain. In monoculture experiments, no significant difference was found between the growth rates or physiological characteristics of the two strains during the exponential growth phase. In contrast, the MT strain was found to dominate the WT strain in co-culture experiments under favorable growth conditions. Moreover, we also found an increase in the growth rate of the MT strain and in the cellular MC content of the WT strain. Our findings suggest that differences in the fitness of these two strains under optimum growth conditions were attributable to the cost to microcystin-producing cells of producing microcystins, and to the putative existence of cooperation processes involving direct interactions between these strains.

Variability of primary and bacterial production in a coral reef lagoon (New Caledonia).

We assessed the temporal variability of nutrients, phytoplankton and bacterioplankton at two sites of different trophic status in New Caledonia's South-West lagoon, a tropical coastal ecosystem. During stable meteorological conditions, Chl.a, bacterial production and nutrient concentrations experience weak but consistent daily variation. Short-term (1-2 week interval) fluctuations of planktonic variables are in the same range as annual variations at both sites. A part of these short term variations is linked to local meteorological events (wind in the main channel, precipitation at the coastal station). Although annual variations are weak compared to short term variations, phytoplankton and bacterioplankton production show consistent temporal patterns, with maxima in December-January and April-May and minima in August. Annual bacterial production represents 21% and 34% of particulate primary production at the oligotrophic and mesotrophic sites, respectively. Mineral nutrient availability indicates that nitrogen is probably the primary limiting factor of phytoplankton throughout the year.

Spatiotemporal changes in the genetic diversity of a bloom-forming Microcystis aeruginosa (cyanobacteria) population.

The variations in microcystin concentrations during cyanobacterial blooms in freshwater ecosystems appear to depend on numerous factors, which have still not been fully identified. To contribute to clarify the situation, we have developed a spatial sampling approach to determine the dynamics and genetic diversity of a bloom-forming population of Microcystis aeruginosa in a large French reservoir, and the variations in the proportions of microcystin-producing genotypes. We demonstrated that marked changes occurred in the internal transcribed spacer (ITS) genotype composition of the M. aeruginosa population during the development of the bloom. These changes led progressively to the selection of one dominant ITS genotype throughout the entire reservoir when the cell number reached its maximum. At the same time, we identified a decrease in the proportion of the mcyB+ genotype, and a significant negative correlation between this proportion and that of the dominant ITS genotype during the bloom. Thus, it appeared that favorable conditions for Microcystis cell growth led to the selection, within the Microcystis population, of a non-microcystin-producing genotype, whereas potentially microcystin-producing genotypes were dominant in this population before and after the bloom, when environmental conditions were less favorable for growth.