Yolanda Del Amo

A novel fluorescent silica tracer for biological silicification studies

BACKGROUND Biological silica production has drawn intense attention and several molecules involved in biosilicification have been identified. Cellular mechanisms, however, remain unknown mainly due to the lack of probes required for obtaining information on live specimens. RESULTS The fluorescence spectra of the compound 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole (PDMPO) are affected by the presence of >3.2 mM silicic acid. Increase in intensity and shift in the fluorescence coincide with the polymerization of Si. The unique PDMPO-silica fluorescence is explored here to visualize Si deposition in living diatoms. The fluorophore is selectively incorporated and co-deposited with Si into the newly synthesized frustules (the outer silica shells) showing an intense green fluorescence. CONCLUSIONS We suggest that a fluorescence shift is due to an interaction between PDMPO and polymeric silicic acid. PDMPO is an excellent probe for imaging newly deposited silica in living cells and has also a potential for a wide range of applications in various Si-related disciplines, including biology of living organisms as diatoms, sponges, and higher plants, clinical research (e.g. lung fibrosis and cancer, bone development, artificial bone implantation), and chemistry and physics of materials research.

THE CHEMICAL FORM OF DISSOLVED SI TAKEN UP BY MARINE DIATOMS

Results of past studies of the pH‐dependent Si uptake kinetics of Phaeodactylum tricornutum Bohlin suggested that the anion SiO(OH) is the chemical form of dissolved Si taken up by marine diatoms. We determined the chemical form of Si taken up by three other marine diatom species and P. tricornutum by examining the kinetics of Si use under two dramatically different SiO(OH):Si(OH)4 ratios in seawater by varying pH from ≈8 to ≈9.6. Uptake rates were determined using a precise and sensitive 32Si tracer methodology. The pH‐dependent uptake kinetics obtained for all species except P. tricornutum suggest that marine diatoms transport Si(OH)4. The half‐saturation constant (Km) varies strongly as a function of pH for all species when the substrate of transport is assumed to be SiO(OH). Kinetic curves for Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, Thalassiosira weissflogii (Grunow) G. Fryxell et Hasle, and Cylindrotheca fusiformis Reimann et Lewin have statistically identical values of Km at each pH when the substrate for transport is assumed to be Si(OH)4 (T. pseudonana and T. weissflogii) or total dissolved silicon (C. fusiformis). In contrast, P. tricornutum exhibits unusual biphasic uptake kinetics: uptake conforms to Michaelis–Menten kinetics up to 15 to 25 μM, above which uptake increases linearly. This enigmatic response may have biased conclusions drawn from past experiments using this species. However, based on the consistency of the results for the three other species, a new model of Si transport in marine diatoms is proposed on the basis of the direct formation of a complex between the Si‐transport protein and Si(OH)4.

Resistance of a coastal ecosystem to increasing eutrophic conditions: the Bay of Brest (France), a semi-enclosed zone of Western Europe

Abstract The Bay of Brest is a semi-enclosed coastal ecosystem receiving high nutrients loading from freshwater inputs. In order to analyse the response of phytoplankton stocks to increasing eutrophic conditions, a survey of the annual cycle of hydrographic properties, nutrients and chlorophyll a concentrations, and carbon uptake rates was performed at four stations in 1993. This database has been compared to earlier measurements performed during several comparable surveys within the last 20 years. As compared to the seventies, a doubled nitrate loading is now entering this ecosystem, which is related to increased agricultural activities on the drainage basins, while the geographical origin of the nitrate input has been modified. As a result of these anthropogenic modifications, summer averaged Si/N stoichiometric balance has decreased during the two last decades but, contrary to what has been observed in other coastal ecosystems, phytoplankton stocks have not increased. Several ecological factors have hindered eutrophication: the high hydrodynamic mixing with adjacent marine waters, caused by the macrotidal regime, induces important nutrients losses, temperature and mostly light limit primary production while Si and P high recycling maintain nitrogen limitation in this ecosystem. Conjunction of these non-anthropogenic factors explains the global stability of phytoplankton stocks.

Pelagic food web patterns: do they modulate virus and nanoflagellate effects on picoplankton during the phytoplankton spring bloom?

As agents of mortality, viruses and nanoflagellates impact on picoplankton populations. We examined the differences in interactions between these compartments in two French Atlantic bays. Microbes, considered here as central actors of the planktonic food web, were first monitored seasonally in Arcachon (2005) and Marennes-Oléron (2006) bays. Their dynamics were evaluated to categorize trophic periods using the models of Legendre and Rassoulzadegan as a reference framework. Microbial interactions were then compared through 48 h batch culture experiments performed during the phytoplankton spring bloom, identified as herbivorous in Marennes and multivorous in Arcachon. Marennes was spatially homogeneous compared with Arcachon. The former was potentially more productive, featuring a large number of heterotrophic pathways, while autotrophic mechanisms dominated in Arcachon. A link was found between viruses and phytoplankton in Marennes, suggesting a role of virus in the regulation of autotroph biomass. Moreover, the virus-bacteria relation was weaker in Marennes, with a bacterial lysis potential of 2.6% compared with 39% in Arcachon. The batch experiments (based on size-fractionation and viral enrichment) revealed different microbial interactions that corresponded to the spring-bloom trophic interactions in each bay. In Arcachon, where there is a multivorous web, flagellate predation and viral lysis acted in an opposite way on picophytoplankton. When together they both reduced viral production. Conversely, in Marennes (herbivorous web), flagellates and viruses together increased viral production. Differences in the composition of the bacterial community composition explained the combined flagellate-virus effects on viral production in the two bays.

Theoretical constraints on the uptake of silicic acid species by marine diatoms

Diatoms, one of the most productive phytoplankton groups in the ocean, have an essential requirement for silicic acid for the construction of their opaline shells. In seawater silicic acid is present in three chemical species (H4SiO4, H3SiO4-, H2SiO42-). The silicic acid molecule actually taken up by diatoms is still under discussion. Algal cells are surrounded by a diffusive boundary layer (DBL) which has an effective thickness of the order of the cell radius. The nutrient transport through this layer is by diffusion only and thus may limit the supply of silicic acid to the cell. Due to uptake of one of the species of silicic acid by the cell, the chemical system in the DBL is out of equilibrium. We have developed a diffusion-reaction model for the components H4SiO4, H3SiO4-, H2SiO42-, OH-, and H+ in the DBL which allows us to calculate maximum silicic acid supply rates as a function of the total concentration of dissolved silicon, pH, algal radius, and silicic acid species taken up by the cell. In addition, analytical solutions for the simplified diffusion-reaction system are presented. Model calculations of the silicic acid maximum uptake rates are compared with uptake data for Thalassiosira weissflogii from recent laboratory experiments. Results indicate that the supply of H4SiO4 to T. weissflogii by diffusion-reaction processes is suffcient to sustain the observed growth rates. Thus, the H4SiO4 uptake of T. weissffogii is not diffusion-limited. This does not hold true for H3SiO4-.

Testing a Microarray to Detect and Monitor Toxic Microalgae in Arcachon Bay in France

Harmful algal blooms (HABs) occur worldwide, causing health problems and economic damages to fisheries and tourism. Monitoring agencies are therefore essential, yet monitoring is based only on time-consuming light microscopy, a level at which a correct identification can be limited by insufficient morphological characters. The project MIDTAL (Microarray Detection of Toxic Algae)—an FP7-funded EU project—used rRNA genes (SSU and LSU) as a target on microarrays to identify toxic species. Furthermore, toxins were detected with a newly developed multiplex optical Surface Plasmon Resonance biosensor (Multi SPR) and compared with an enzyme-linked immunosorbent assay (ELISA). In this study, we demonstrate the latest generation of MIDTAL microarrays (version 3) and show the correlation between cell counts, detected toxin and microarray signals from field samples taken in Arcachon Bay in France in 2011. The MIDTAL microarray always detected more potentially toxic species than those detected by microscopic counts. The toxin detection was even more sensitive than both methods. Because of the universal nature of both toxin and species microarrays, they can be used to detect invasive species. Nevertheless, the MIDTAL microarray is not completely universal: first, because not all toxic species are on the chip, and second, because invasive species, such as Ostreopsis, already influence European coasts.

Microfluidic technology for plankton research

Plankton produces numerous chemical compounds used in cosmetics and functional foods. They also play a key role in the carbon budget on the Earth. In a context of global change, it becomes important to understand the physiological response of these microorganisms to changing environmental conditions. Their adaptations and the response to specific environmental conditions are often restricted to a few active cells or individuals in large populations. Using analytical capabilities at the subnanoliter scale, microfluidic technology has also demonstrated a high potential in biological assays. Here, we review recent advances in microfluidic technologies to overcome the current challenges in high content analysis both at population and the single cell level.

High-Content Screening of Plankton Alkaline Phosphatase Activity in Microfluidics

One way for phytoplankton to survive orthophosphate depletion is to utilize dissolved organic phosphorus by expressing alkaline phosphatase. The actual methods to assay alkaline phosphate activity-either in bulk or as a presence/absence of enzyme activity-fail to provide information on individual living cells. In this context, we develop a new microfluidic method to compartmentalize cells in 0.5 nL water-in-oil droplets and measure alkaline phosphatase activity at the single-cell level. We use enzyme-labeled fluorescence (ELF), which is based on the hydrolysis of ELF-P substrate, to monitor in real time and at the single-cell level both qualitative and quantitative information on cell physiology (i.e., localization and number of active enzyme sites and alkaline phosphatase kinetics). We assay the alkaline phosphatase activity of Tetraselmis sp. as a function of the dissolved inorganic phosphorus concentration and show that the time scale of the kinetics spans 1 order of magnitude. The advantages of subnanoliter-scale compartmentalization in droplet-based microfluidics provide a precise characterization of a population with single-cell resolution. Our results highlight the key role of cell physiology to efficiently access dissolved organic phosphorus.