The Transition Toward Nitrogen Deprivation in Diatoms Requires Chloroplast Stand-By and Deep Metabolic Reshuffling.

Microalgae have adapted to face abiotic stresses by accumulating energy storage molecules such as lipids, which are also of interest to industries. Unfortunately, the impairment in cell division during the accumulation of these molecules constitutes a major bottleneck for the development of efficient microalgae-based biotechnology processes. To address the bottleneck, a multidisciplinary approach was used to study the mechanisms involved in the transition from nitrogen repletion to nitrogen starvation conditions in the marine diatom Phaeodactylum tricornutum that was cultured in a turbidostat. Combining data demonstrate that the different steps of nitrogen deficiency clustered together in a single state in which cells are in equilibrium with their environment. The switch between the nitrogen-replete and the nitrogen-deficient equilibrium is driven by intracellular nitrogen availability. The switch induces a major gene expression change, which is reflected in the reorientation of the carbon metabolism toward an energy storage mode while still operating as a metabolic flywheel. Although the photosynthetic activity is reduced, the chloroplast is kept in a stand-by mode allowing a fast resuming upon nitrogen repletion. Altogether, these results contribute to the understanding of the intricate response of diatoms under stress.

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.

Metabolomics based on UHPLC-QToF- and APGC-QToF-MS reveals metabolic pathways reprogramming in response to tidal cycles in the sub-littoral species Mimachlamys varia exposed to aerial emergence.

Mimachlamys varia is a sub-littoral bivalve encountered from Norway to the Mediterranean Sea, which lives mostly byssally attached to rocks. During the low tide period, M. varia individuals, located highest on the shore, may experience short time of aerial exposure and face a low availability of oxygen. Here we report a comparative metabolomic profiling of gill samples of M. varia obtained by both LC-QToF and APGC-QToF mass spectrometry, to analyze metabolic changes occurring during emersion in comparison with immersion. Scallops were grown in aquaria with a simulated intertidal environment mimicking short-duration air exposure that they might experience during extreme tides: alternating 2 h emersion and 10 h immersion. Our results show a switch from aerobic to anaerobic metabolism after only 2 h of emersion, with the resort to different pathways: glucose-lactate, glucose-succinate and aspartate-succinate pathways. Furthermore, carnitine-conjugated metabolites were found to accumulate during emersion, as well as urate. The level of tyrosine on the contrary was found to decrease. These findings indicate a complex metabolic reprogramming that occurs after a two hour emersion period and upon re-immersion. Furthermore, M. varia is used as sentinel species in pollution biomonitoring, through the assay of biomarkers to evaluate the effects of pollutants. Here we show that emersion induces a significant decrease of superoxide dismutase activity, an enzyme developed by bivalves to face oxidative stress and used as biomarker. These findings have to be taken into account to normalize sampling during campaigns of environmental monitoring, by taking in situ, as far as possible only immersed individuals.

Identification and separation of saxitoxins using hydrophilic interaction liquid chromatography coupled to traveling wave ion mobility-mass spectrometry.

The aim of this work was to develop a reliable and efficient analytical method to characterise and differentiate saxitoxin analogues (STX), including sulphated (gonyautoxins, GTX) and non-sulphated analogues. For this purpose, hydrophilic interaction liquid chromatography (HILIC) was used to separate sulphated analogues. We also resorted to ion mobility spectrometry to differentiate the STX analogues because this technique adds a new dimension of separation based on ion gas phase conformation. Positive and negative ionisation modes were used for gonyautoxins while positive ionisation mode was used for non-sulphated analogues. Subsequently, the coupling of these three complementary techniques, HILIC-IM-MS, permitted the separation and identification of STX analogues; isomer differentiation was achieved in HILIC dimension while non-sulphated analogues were separated in the IM-MS dimension. Additional structural characteristics concerning the conformation of STXs could be obtained using IM-MS measurements. Thus, the collision cross sections (CCS) of STXs are reported for the first time in the positive ionisation mode. These experimental CCSs correlated well with the calculated CCS values using the trajectory method.

Extended evaluation of polymeric and lipophilic sorbents for passive sampling of marine toxins.

Marine biotoxins are algal metabolites that can accumulate in fish or shellfish and render these foodstuffs unfit for human consumption. These toxins, released into seawater during algal occurrences, can be monitored through passive sampling. Acetone, methanol and isopropanol were evaluated for their efficiency in extracting toxins from algal biomass. Isopropanol was chosen for further experiments thanks to a slightly higher recovery and no artifact formation. Comparison of Oasis HLB, Strata-X, BondElut C18 and HP-20 sorbent materials in SPE-mode led to the choice of Oasis HLB, HP-20 and Strata-X. These three sorbents were separately exposed as passive samplers for 24 h to seawater spiked with algal extracts containing known amounts of okadaic acid (OA), azaspiracids (AZAs), pinnatoxin-G (PnTX-G), 13-desmethyl spirolide-C (SPX1) and palytoxins (PlTXs). Low density polyethylene (LDPE) and silicone rubber (PDMS) strips were tested in parallel on similar mixtures of spiked natural seawater for 24 h. These strips gave significantly lower recoveries than the polymeric sorbents. Irrespective of the toxin group, the adsorption rate of toxins on HP-20 was slower than on Oasis HLB and Strata-X. However, HP-20 and Strata-X gave somewhat higher recoveries after 24 h exposure. Irrespective of the sorbent tested, recoveries were generally highest for cyclic imines and OA group toxins, slightly lower for AZAs, and the lowest for palytoxins. Trials in re-circulated closed tanks with mussels exposed to Vulcanodinium rugosum or Prorocentrum lima allowed for further evaluation of passive samplers. In these experiments with different sorbent materials competing for toxins in the same container, Strata-X accumulated toxins faster than Oasis HLB, and HP-20, and to higher levels. The deployment of these three sorbents at Ingril French Mediterranean lagoon to detect PnTX-G in the water column showed accumulation of higher levels on HP-20 and Oasis HLB compared to Strata-X. This study has significantly extended the range of sorbents for passive sampling of marine toxins. In particular, sorbents were included that had previously been evaluated for polyhalogenated contaminants, pharmaceuticals, phytochemicals or veterinary residues. Moreover, this study has for the first time demonstrated the usefulness of the polymeric Oasis HLB and Strata-X sorbents in laboratory and field studies for various microalgal toxins.

Cytotoxicity, fractionation and dereplication of extracts of the dinoflagellate Vulcanodinium rugosum, a producer of pinnatoxin G.

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of "fast-acting toxins", its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC50 values of 0.38 µg mL⁻¹ and 0.19 µg mL⁻¹ were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL⁻¹). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.

An integrated environmental approach to investigate biomarker fluctuations in the blue mussel Mytilus edulis L. in the Vilaine estuary, France.

Estuarine areas represent complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. Therefore, the aquatic organisms living in estuaries have to face highly variable environmental conditions. The aim of this work was to study the influence of environmental changes from either natural or anthropogenic origins on the physiological responses of Mytilus edulis. Mussels were collected in the Vilaine estuary during early summer because this season represents a critical period of active reproduction in mussels and of increased anthropogenic inputs from agricultural and boating activities into the estuary. The physiological status of the mussel M. edulis was evaluated through measurements of a suite of biomarkers related to: oxidative stress (catalase, malondialdehyde), detoxication (benzopyrene hydroxylase, carboxylesterase), neurotoxicity (acetylcholinesterase), reproductive cycle (vitelline, condition index, maturation stages), immunotoxicity (hemocyte concentration, granulocyte percentage, phagocytosis, reactive oxygen species production, oxidative burst), and general physiological stress (lysosomal stability). A selection of relevant organic contaminant (pesticides, (polycyclic aromatic hydrocarbons, polychlorobiphenyls) was measured as well as environmental parameters (water temperature, salinity, total suspended solids, turbidity, chlorophyll a, pheopigments) and mussel phycotoxin contamination. Two locations differently exposed to the plume of the Vilaine River were compared. Both temporal and inter-site variations of these biomarkers were studied. Our results show that reproduction cycle and environmental parameters such as temperature, organic ontaminants, and algal blooms could strongly influence the biomarker responses. These observations highlight the necessity to conduct integrated environmental approaches in order to better understand the causes of biomarker variations.

Determination of peptaibol trace amounts in marine sediments by liquid chromatography/electrospray ionization-ion trap-mass spectrometry.

Extraction followed by reverse phase liquid chromatography (LC)/electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS) analysis has been successfully developed for the determination of peptaibols, fungal toxic metabolites, in marine sediments. Spiking experiments showed that the mean recovery of target compounds exceeded 85% at a spiking level of 10 ng/g of sediment (wet weight). Detection and quantification limits were 250 and 830 pg/g of sediment, respectively. The method developed constituted the first sensitive assay for quantification of peptaibol trace amounts in a natural environment. A concentration of 5 ng/g in sediment samples collected from Fier d'Ars was found.

New short peptaibols from a marine Trichoderma strain.

The production of peptaibols by a marine-related Trichoderma longibrachiatum strain was studied using electrospray ionisation multiple-stage ion trap mass spectrometry (ESI-MSn-IT) and gas chromatography/electron impact mass spectrometry (GC/EI-MS). Two major groups of peptaibols were identified, those with long sequences (20 amino acids) and others with short sequences (11 amino acids). This paper describes the methodology used to establish the sequences of short peptaibols in a mixture without previous individual separation. Nine peptaibols were identified. Among them, eight are new, namely as trichobrachin A I-IV (Aib9-Pro10 sequence) and as trichobrachin B I-IV (Val9-Pro10 sequence). Original Pro6-Val7 and Val9-Pro10 sequences have to be noted.

Combined use of LC/MS and a biological test for rapid identification of marine mycotoxins produced by Trichoderma koningii.

Trichoderma koningii Oudemans, a strain isolated from a shellfish farming area, was selected for its high frequency in samples and its ability to produce metabolites when cultured in natural seawater. Combined use of LC/MS and a biological test on blowfly larvae allowed the characterization of four compounds after purification in only two steps (VLC and HPLC). ESI/MS, a powerful tool for rapid identification and sequence determination of peptides, confirmed that these compounds were peptide, alpha-aminoisobutyric acid and amino alcohol (peptaibols), the usual metabolites of Trichoderma.