Influence of Sharklet-Inspired Micropatterned Polymers on Spatio-Temporal Variations of Marine Biofouling.

This article aims to show the influence of surface characteristics (microtopography, chemistry, mechanical properties) and seawater parameters on the settlement of marine micro- and macroorganisms. Polymers with nine microtopographies, three distinct mechanical properties, and wetting characteristics are immersed for one month into two contrasting coastal sites (Toulon and Kristineberg Center) and seasons (Winter and Summer). Influence of microtopography and chemistry on wetting is assessed through static contact angle and captive air bubble measurements over 3-weeks immersion in artificial seawater. Microscopic analysis, quantitative flow cytometry, metabarcoding based on the ribulose biphosphate carboxylase (rbcL) gene amplification, and sequencing are performed to characterize the settled microorganisms. Quantification of macrofoulers is done by evaluating the surface coverage and the type of organism. It is found that for long static in situ immersion, mechanical properties and non-evolutive wettability have no major influence on both abundance and diversity of biofouling assemblages, regardless of the type of organisms. The apparent contradiction with previous results, based on model organisms, may be due to the huge diversity of marine environments, both in terms of taxa and their size. Evolutive wetting properties with wetting switching back and forth over time have shown to strongly reduce the colonization by macrofoulers.

Impact of phosphate concentration on the metabolome of biofilms of the marine bacterium Pseudoalteromonas lipolytica.

INTRODUCTION: Marine biofilms are the most widely distributed mode of life on Earth and drive biogeochemical cycling processes of most elements. Phosphorus (P) is essential for many biological processes such as energy transfer mechanisms, biological information storage and membrane integrity. OBJECTIVES: Our aim was to analyze the effect of a gradient of ecologically relevant phosphate concentrations on the biofilm-forming capacity and the metabolome of the marine bacterium Pseudoalteromonas lipolytica TC8. METHODS: In addition to the evaluation of the effect of different phosphate concentration on the biomass, structure and gross biochemical composition of biofilms of P. lipolytica TC8, untargeted metabolomics based on liquid chromatography-mass spectrometry (LC-MS) analysis was used to determine the main metabolites impacted by P-limiting conditions. Annotation of the most discriminating and statistically robust metabolites was performed through the concomitant use of molecular networking and MS/MS fragmentation pattern interpretation. RESULTS: At the lowest phosphate concentration, biomass, carbohydrate content and three-dimensional structures of biofilms tended to decrease. Furthermore, untargeted metabolomics allowed for the discrimination of the biofilm samples obtained at the five phosphate concentrations and the highlighting of a panel of metabolites mainly implied in such a discrimination. A large part of the metabolites of the resulting dataset were then putatively annotated. Ornithine lipids were found in increasing quantity when the phosphate concentration decreased, while the opposite trend was observed for oxidized phosphatidylethanolamines (PEs). CONCLUSION: This study demonstrated the suitability of LC-MS-based untargeted metabolomics for evaluating the effect of culture conditions on marine bacterial biofilms. More precisely, these results supported the high plasticity of the membrane of P. lipolytica TC8, while the role of the oxidized PEs remains to be clarified.

Seawater copper content controls biofilm bioaccumulation and microbial community on microplastics.

The adsorption of trace metals on microplastics (MPs) is affected by the presence of surficial biofilms but their interactions are poorly understood. Here, we present the influence of Cu levels in real seawater (Toulon Bay, NW Mediterranean Sea) on microbial communities and Cu content of the resulting biofilms grown during incubation experiments on high density polyethylene. Two sets of incubation experiments were run with seawater supplied with MPs, sampled in two sites with contrasting Cu levels: Pt12 (most contaminated site) and Pt41P (less contaminated site). For each incubation experiment, 5 treatments were considered differing in Cu concentrations, ranging between 30 and 400 nM and between 6 and 60 nM, for Pt12 and Pt41p, respectively. A control experiment (filtered at 0.2 µm) was run in parallel for each incubation experiment. We observed that, at the time scale of the incubation period, both prokaryotic and eukaryotic richness and diversity were higher in the biofilms formed from the most contaminated site. In addition, we showed that Cu levels are shaping biofilm communities, evidencing co-occurrence patterns between prokaryotes and eukaryotes with diatoms playing a central role. These differences in biofilm formation were reflected in the amount of bioaccumulated Cu per dry weight of MPs, exhibiting higher values in the most contaminated site. Within this site, the increase of Cu seawater content enhanced its bioaccumulation onto MPs until reaching saturation. This study strongly suggests a striking link between seawater copper content, biofilm community shaping and the resulting Cu bioaccumulation onto MPs.

Temperate and tropical coastal waters share relatively similar microbial biofilm communities while free-living or particle-attached communities are distinct.

Free-living (FL) marine microbial communities differ from those attached to particles (PA). Likewise, biofilms (B) colonizing artificial surfaces, including plastics or ship hulls, hardly resemble their planktonic surroundings. However, few studies have examined the effect of the environment on these lifestyles and on the source of organisms colonizing marine surfaces. Using 16S rRNA gene metabarcoding, we identified specificities of marine prokaryotic community lifestyles (FL, PA or B) sampled in three coastal polluted locations with dissimilar environmental conditions: the North-Western Mediterranean Sea and the Atlantic and Indian Oceans. Biofilms developed over polyvinyl chloride (PVC) were found to be significantly different from FL or PA collected during the immersions. Alpha-diversity increased from FL to PA and to B, illustrating the integrative aspect of the latter, with little proportion of operational taxonomic units shared with the first two. Beta-diversity clustered first the lifestyles and then the sites. FL and PA were more affected by water quality, especially by trace metal contamination, whereas B were as sensitive to trace metals as to nutrients. Although biofilms should be supplied by the planktonic (ultra) rare biosphere, source tracking could only detect small contributions of FL or PA taxa to B communities.

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

Antifouling Activities of Immobilized Ferrocenyl Glucose on a Glassy Carbon Surface.

Polar coatings are used to protect surfaces from marine fouling based on the formation of a hydrated surface layer, which acts as a barrier to marine microorganisms. In this context, we have developed a material with glucose bound to ferrocene to prevent surface absorption. Glucose brings the polarity while ferrocene has the role of varying the state of charge of the surface. We therefore describe the synthesis of 6-deoxy-6-(4-ferrocenyl-1H-1,2,3-triazol-1-yl)-1-(4-aminophenyl)-ß-D-glucopyranose 6 and its immobilization on the surface of a C electrode and develop a methodology used for antibacterial testing. We were able to demonstrate that the immobilization of glucose 6 could be done in an artificial seawater environment by oxidation of an amine. The use of a 96-microwell platform equipped with electrodes for cyclic voltammetry, linked to a potentiostat, allowed the electrical solicitation of the coating in the presence of marine bacteria with a greater number of biological replicates. We have shown that the coating has an antibacterial effect, and this effect is accentuated when the coating is electrically stressed, resulting in the appearance and disappearance of charge on ferrocene, a phenomenon that seems to be conducive to colonization by bacteria.

Direct Readout of Neural Stem Cell Transgenesis with an Integration-Coupled Gene Expression Switch.

Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.

A Multi-Omics Analysis Suggests Links Between the Differentiated Surface Metabolome and Epiphytic Microbiota Along the Thallus of a Mediterranean Seaweed Holobiont.

Marine macroalgae constitute an important living resource in marine ecosystems and complex ecological interactions occur at their surfaces with microbial communities. In this context, the present study aimed to investigate how the surface metabolome of the algal holobiont Taonia atomaria could drive epiphytic microbiota variations at the thallus scale. First, a clear discrimination was observed between algal surface, planktonic and rocky prokaryotic communities. These data strengthened the hypothesis of an active role of the algal host in the selection of epiphytic communities. Moreover, significant higher epibacterial density and α-diversity were found at the basal algal parts compared to the apical ones, suggesting a maturation gradient of the community along the thallus. In parallel, a multiplatform mass spectrometry-based metabolomics study, using molecular networking to annotate relevant metabolites, highlighted a clear chemical differentiation at the algal surface along the thallus with similar clustering as for microbial communities. In that respect, higher amounts of sesquiterpenes, phosphatidylcholines (PCs), and diacylglycerylhydroxymethyl-N,N,N-trimethyl-ß-alanines (DGTAs) were observed at the apical regions while dimethylsulfoniopropionate (DMSP) and carotenoids were predominantly found at the basal parts of the thalli. A weighted UniFrac distance-based redundancy analysis linking the metabolomics and metabarcoding datasets indicated that these surface compounds, presumably of algal origin, may drive the zonal variability of the epibacterial communities. As only few studies were focused on microbiota and metabolome variation along a single algal thallus, these results improved our understanding about seaweed holobionts. Through this multi-omics approach at the thallus scale, we suggested a plausible scenario where the chemical production at the surface of T. atomaria, mainly induced by the algal physiology, could explain the specificity and the variations of the surface microbiota along the thallus.

Shear Stress as a Major Driver of Marine Biofilm Communities in the NW Mediterranean Sea.

While marine biofilms depend on environmental conditions and substrate, little is known about the influence of hydrodynamic forces. We tested different immersion modes (dynamic, cyclic and static) in Toulon Bay (north-western Mediterranean Sea; NWMS). The static mode was also compared between Toulon and Banyuls Bays. In addition, different artificial surfaces designed to hamper cell attachment (self-polishing coating: SPC; and fouling-release coating: FRC) were compared to inert plastic. Prokaryotic community composition was affected by immersion mode, surface characteristics and site. Rhodobacteriaceae and Flavobacteriaceae dominated the biofilm community structure, with distinct genera according to surface type or immersion mode. Cell density increased with time, greatly limited by hydrodynamic forces, and supposed to delay biofilm maturation. After 1 year, a significant impact of shear stress on the taxonomic structure of the prokaryotic community developed on each surface type was observed. When surfaces contained no biocides, roughness and wettability shaped prokaryotic community structure, which was not enhanced by shear stress. Conversely, the biocidal effect of SPC surfaces, already major in static immersion mode, was amplified by the 15 knots speed. The biofilm community on SPC was 60% dissimilar to the biofilm on the other surfaces and was distinctly colonized by Sphingomonadaceae ((Alter)Erythrobacter). At Banyuls, prokaryotic community structures were more similar between the four surfaces tested than at Toulon, due possibly to a masking effect of environmental constraints, especially hydrodynamic, which was greater than in Toulon. Finally, predicted functions such as cell adhesion confirmed some of the hypotheses drawn regarding biofilm formation over the artificial surfaces tested here.

96X Screen-Printed Gold Electrode Platform to Evaluate Electroactive Polymers as Marine Antifouling Coatings.

Several alternatives are currently investigated to prevent and control the natural process of colonization of any seawater submerged surfaces by marine organisms. Since few years we develop an approach based on addressable electroactive coatings containing conducting polymers or polymers with lateral redox groups. In this article we describe the use of a screen-printed plate formed by 96 three-electrode electrochemical cells to assess the potential of these electroactive coatings to prevent the adhesion of marine bacteria. This novel platform is intended to control and record the redox properties of the electroactive coating in each well during the bioassay (15 h) and to allow screening its antiadhesion activity with enough replicates to support significant conclusions. Validation of this platform was carried out with poly(ethylenedioxythiophene) (PEDOT) as electroactive coating obtained by electropolymerization of EDOT monomer in artificial seawater electrolyte on the working electrode of each electrochemical cell of the 96-well microplate.