Use of the dummy approach for the synthesis of ion imprinted polymers with Ni(II) or Zn(II) as template ion for the solid-phase extraction of Cu(II).

There is a strong interest in monitoring copper in environmental waters, but its direct analysis suffers from strong matrix interferences. This is why, a sample pretreatment based on solid-phase extraction (SPE) is often used but conventional sorbents usually lack specificity. It is overcome with ion-imprinted polymers (IIPs). This work evaluates for the first time the use of the dummy approach for the synthesis of Cu(II)-targeting IIPs. Two analog ions Ni(II) and Zn(II) were tested as templates and the resulting IIPs were packed in SPE cartridges. The SPE procedure was designed by optimizing a washing step following the sample percolation, to eliminate the interfering ions retained on the IIP by non-specific interactions. To optimize the washing step, solutions at different pH or containing tris(hydroxymethyl)aminomethane as a complexing agent at different concentrations were tested and combined. Zn-IIP appeared more promising than Ni-IIP, showing excellent specificity and a high selectivity. Its retention capacity was determined to be 100 µg/g, and different isotherm models were evaluated to fit with the adsorption data. Finally, applications to mineral and sea waters were successfully completed and led to high and repeatable extraction recoveries for Cu(II) (88 ± 1% and 83 ± 3%, respectively).

Evaluation of lipophosphoramidates-based amphiphilic compounds on the formation of biofilms of marine bacteria.

The bacteriostatic and/or bactericidal properties of few phosphoramide-based amphiphilic compounds on human pathogenic bacteria were previously reported. In this study, the potential of two cationic (BSV36 and KLN47) and two zwitterionic (3 and 4) amphiphiles as inhibitors of marine bacterial growth and biofilm formation were investigated. Results showed that the four compounds have little impact on the growth of a panel of 18 selected marine bacteria at a concentration of 200 µM, and up to 700 µM for some bacterial strains. Interestingly, cationic lipid BSV36 and zwitterionic lipids 3 and 4 effectively disrupt biofilm formation of Paracoccus sp. 4M6 and Vibrio sp. D02 at 200 µM and to a lesser extent of seven other bacterial strains tested. Moreover, ecotoxicological assays on four species of microalgae highlighted that compounds 3 and 4 have little impact on microalgae growth with EC50 values of 51 µM for the more sensitive species and up to 200 µM for most of the others. Amphiphilic compounds, especially zwitterionic amphiphiles 3 and 4 seem to be promising candidates against biofilm formation by marine bacteria.

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria.

Biofilms of heterotrophic bacteria cover organic matter aggregates and constitute hotspots of mineralization, primarily acting through extracellular hydrolytic enzyme production. Nevertheless, regulation of both biofilm and hydrolytic enzyme synthesis remains poorly investigated, especially in estuarine ecosystems. In this study, various bioassays, mass spectrometry and genomics approaches were combined to test the possible involvement of quorum sensing (QS) in these mechanisms. QS is a bacterial cell-cell communication system that relies notably on the emission of N-acylhomoserine lactones (AHLs). In our estuarine bacterial collection, we found that 28 strains (9%), mainly Vibrio, Pseudomonas and Acinetobacter isolates, produced at least 14 different types of AHLs encoded by various luxI genes. We then inhibited the AHL QS circuits of those 28 strains using a broad-spectrum lactonase preparation and tested whether biofilm production as well as ß-glucosidase and leucine-aminopeptidase activities were impacted. Interestingly, we recorded contrasted responses, as biofilm production, dissolved and cell-bound ß-glucosidase and leucine-aminopeptidase activities significantly increased in 4%-68% of strains but decreased in 0%-21% of strains. These findings highlight the key role of AHL-based QS in estuarine bacterial physiology and ultimately on biogeochemical cycles. They also point out the complexity of QS regulations within natural microbial assemblages.

Towards the understanding of the uptake and depuration of microplastics in the ragworm Hediste diversicolor: Field and laboratory study.

An important number of studies have evaluated the presence of microplastics, particles with a size below 5 mm, in aquatic organisms. Studies have shown that these fragments are widely present in the marine environment, but research on the estuarine ecosystem is still scarce. In this study, two different approaches were used to evaluate the presence and ingestion of plastic particles in the ragworm Hediste diversicolor: a field study for the environmental assessment and a laboratory experiment in controlled condition. For the environmental evaluation, ingestion of microplastics was evaluated in the ragworm H. diversicolor sampled from the mudflats of the Seine estuary (France) during March and June 2017 and 2018, on two locations: S1 and S2, both characterized by high anthropogenic pressures, and for S2 a more influential hydrodynamic component. Ingestion of microplastics was measured in ragworms tissues and in gut content (sediment) after depuration. The number of particles as well as their size, shape and color were reported and compared between sampling period and locations. Results showed the presence of a low number of particles in both worms and gut content. In gut content, 45.6% and 87.58% of samples from site S1 and S2 respectively contained plastic like particles. In worms, 41.7% (S1) and 75.8% (S2) of analysed samples contained plastic like items. The lowest mean number of particles was 0.21 ± 0.31 (S1 in June 2017) in worms' tissues, but 0.80 ± 0.90 (S1 in June 2017) in the gut content and the highest was 1.47 ± 1.41 (S2 in April 2017) while the highest number was 2.55 ± 2.06 (S2 in June 2017) in worms and gut content respectively. The majority of suspected microplastics observed were fibers (66%) and fragments (27%), but films (3.7%) foam (2.1%), and granules (0.2%) were also identified. In addition, the most polymer type observed by Raman spectroscopy was polypropylene. Furthermore, a preliminary study of the ingestion and egestion of fluorescent polyethylene (PE) microbeads in the digestive tract of ragworms was conducted after exposure through water, during 1h at 1.2 × 106 MP/mL. Results showed a rapid turnover of PE microbeads throughout the digestive tract of worms especially after exposure through water. This study revealed that microplastics are ingested by the ragworm H. diversicolor but do not seem to bioaccumulate. More research is needed to measure potential chronic effects of microplastics on physiological parameters of H. diversicolor and potential trophic transfer of microplastics.

Development of ion-imprinted polymers for the selective extraction of Cu(II) ions in environmental waters.

Several ion-imprinted polymers (IIPs) were synthesized via bulk polymerization with Cu(II) as template ion, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinking agent, and azobisisobutyronitrile as initiator in acetonitrile or methanol as porogen solvent. Non-imprinted polymers (NIPs) were similarly synthesized but without Cu(II). After grounding and sieving, the template ions were removed from IIPs particles through several cycles of elimination in 3 M HCl. All NIPs were equally subjected to this acid treatment with the exception of one NIP, called unwashed NIP. The resulting IIP/NIP particles were packed in solid phase extraction (SPE) cartridges for characterization. The SPE protocol was designed by optimizing a washing step following the sample percolation to eliminate potential interfering ions prior to the elution of Cu(II), all fractions analyzed by inductively coupled plasma mass spectrometry. The best IIP showed a high specificity (recovery of Cu(II) vs. interfering ions) and a good selectivity (retention on IIP vs. NIP). Its adsorption capacity was determined to be 63 µg g-1. Then, a volume of 50 mL was percolated with 30 mg of IIP, thus giving rise to an enrichment factor of 24. Finally, applications to real samples (mineral and sea waters) were successfully performed. In addition, Brunauer-Emmett-Teller analyses showed that the surface area of the washed NIP was almost double that of the unwashed one (140.70 vs. 74.49 m2 g-1), demonstrating for the first time that the post-treatment of a NIP after its synthesis may have a significant impact on its porous structure, and thus need to be more precisely detailed by authors in the future papers.

Wirelessly Powered Drug-Free and Anti-Infective Smart Bandage for Chronic Wound Care.

We present a wirelessly powered ultraviolet-C (UVC) radiation-based disinfecting bandage for sterilization and treatment in chronic wound care and management. The bandage contains embedded low-power UV light-emitting diodes (LEDs) in the 265 to 285 nm range with the light emission controlled via a microcontroller. An inductive coil is seamlessly concealed in the fabric bandage and coupled with a rectifier circuit to enable 6.78 MHz wireless power transfer (WPT). The maximum WPT efficiency of the coils is 83% in free space and 75% on the body at a coupling distance of 4.5 cm. Measurements show that the UVC LEDs are emitting radiant power of about 0.6 mW and 6.8 mW with and without fabric bandage, respectively, when wirelessly powered. The ability of the bandage to inactivate microorganisms was examined in a laboratory which shows that the system can effectively eradicate Gram-negative bacteria, Pseudoalteromonas sp. D41 strain, on surfaces in six hours. The proposed smart bandage system is low-cost, battery-free, flexible and can be easily mounted on the human body and, therefore, shows great promise for the treatment of persistent infections in chronic wound care.

Proteomic studies highlight outer-membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41.

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer-membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer-membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB-dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT-PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.

Quorum Sensing Regulates the Hydrolytic Enzyme Production and Community Composition of Heterotrophic Bacteria in Coastal Waters.

Heterotrophic microbial communities play a central role in biogeochemical cycles in the ocean by degrading organic matter through the synthesis of extracellular hydrolytic enzymes. Their hydrolysis rates result from the community's genomic potential and the differential expression of this genomic potential. Cell-cell communication pathways such as quorum sensing (QS) could impact both aspects and, consequently, structure marine ecosystem functioning. However, the role of QS communications in complex natural assemblages remains largely unknown. In this study, we investigated whether N-acylhomoserine lactones (AHLs), a type of QS signal, could regulate both hydrolytic activities and the bacterial community composition (BCC) of marine planktonic assemblages. To this extent, we carried out two microcosm experiments, adding five different AHLs to bacterial communities sampled in coastal waters (during early and peak bloom) and monitoring their impact on enzymatic activities and diversity over 48 h. Several specific enzymatic activities were impacted during both experiments, as early as 6 h after the AHL amendments. The BCC was also significantly impacted by the treatments after 48 h, and correlated with the expression of the hydrolytic activities, suggesting that changes in hydrolytic intensities may drive changes in BCC. Overall, our results suggest that QS communication could participate in structuring both the function and diversity of marine bacterial communities.

Microplastics contamination in pearl-farming lagoons of French Polynesia.

Pearl-farming is the second most important source of income in French Polynesia. However, tropical lagoons are fragile ecosystems with regard to anthropogenic pressures like plastic pollution, which threaten marine life and the pearl oyster-related economy. Here, we investigated the spatial distribution of microplastics (MP) and concentrations in surface water (SW), water column (WC) and cultivated pearl oyster (PO) from three pearl-farming atolls with low population and tourism. Microplastics were categorized by their size class, shape, colour and polymer type identified using FTIR spectroscopy. Widespread MP contamination was observed in every study site (SW, 0.2-8.4 MP m-3; WC, 14.0-716.2 MP m-3; PO, 2.1-125.0 MP g-1 dry weight), with high contamination in the WC highlighting the need to study the vertical distribution of MP, especially as this compartment where PO are reared. A large presence of small (< 200 µm) and fragment-shaped (> 70%) MP suggests that they result from the breakdown of larger plastic debris. The most abundant polymer type was polyethylene in SW (34-39%), WC (24-32%), while in PO, polypropylene (14-20%) and polyethylene were more evenly distributed (9-21%). The most common MP identified as black-grey polyethylene and polypropylene matches the polymer and colour of ropes and collectors questioning a pearl-farming origin.

Marine fish spermatozoa: racing ephemeral swimmers.

After a long period of spermatogenesis (several weeks to months), marine fish spermatozoa are delivered at male spawning in seawater (SW) at the same time as ova. In some fish species, as the ova micropyle closes quickly after release, these minute unicells, the spermatozoa, have to accomplish their task of reaching the micropyle within a very brief period (several seconds to minutes), for delivery of the haploid male genetic information to the ova. To achieve this goal, their high-performance motile equipment, the flagellum, must fully activate immediately on contact with the SW and then propel the sperm cell at an unusually high initial velocity. The cost of such 'hyperactivity' is a very rapid consumption of intracellular ATP that outstrips the supply. The spermatozoa become rapidly exhausted because mitochondria cannot compensate for this very fast flagellar energy consumption. Therefore, any spermatozoon ends up with two possibilities: either becoming exhausted and immotile or reaching the egg micropyle within its very short period of forward motility (in the range of tens of seconds) before micropyle closure in relation to both contact of SW and cortical reaction. The aim of the present review is to present step by step the successive events occurring in marine fish spermatozoa from activation until their full arrest of motility. The present knowledge of activation mechanisms is summarized, as well as a description of the motility parameters characterizing the motility period. As a complement, in vitro results on axonemal motility obtained after demembranation of flagella bring further understanding. The description of the sperm energetic content (ATP and other high energy compounds) and its evolution during the swimming period is also discussed. A general model aiming to explain all the successive cellular events occurring immediately after the activation is presented. This model is proposed as a guideline for understanding the events governing the sperm lifespan in the marine fish species that reproduce through external fertilization.

Locating the barnacle settlement pheromone: spatial and ontogenetic expression of the settlement-inducing protein complex of Balanus amphitrite.

Barnacles are prominent members of hard substratum benthic communities and their study has been important to advances in experimental ecology and contemporary ecological theory. Having recently characterized the cue to gregarious settlement of Balanus amphitrite, the settlement-inducing protein complex (SIPC), we use two polyclonal antibodies to examine the tissue distribution and ontogenetic expression of this glycoprotein. These antibodies were raised against two separate peptides located near the N- and C-termini of the SIPC and were used to detect the glycoprotein by western blotting and immunohistochemistry. By in situ hybridization we also show that the SIPC mRNA co-occurs with the expressed glycoprotein in the cuticles of both nauplius and cypris larval stages and the adult. In the larvae, the SIPC is expressed most strongly in the mouthparts and the hindgut of the stage 2 nauplius and in the thoracopods, antennules and bivalved carapace of the cyprid. In adult B. amphitrite, the expressed SIPC is present in protein extracts of the shell and in all organs that are lined by cuticular tissues. We suggest that the SIPC is produced by the epidermal cells that secrete the cuticle and discuss these observations with regard to earlier studies and the role of the SIPC as a contact pheromone.

Rapid detection and quantification of the marine toxic algae, Alexandrium minutum, using a super-paramagnetic immunochromatographic strip test.

The dinoflagellates of Alexandrium genus are known to be producers of paralytic shellfish toxins that regularly impact the shellfish aquaculture industry and fisheries. Accurate detection of Alexandrium including Alexandrium minutum is crucial for environmental monitoring and sanitary issues. In this study, we firstly developed a quantitative lateral flow immunoassay (LFIA) using super-paramagnetic nanobeads for A. minutum whole cells. This dipstick assay relies on two distinct monoclonal antibodies used in a sandwich format and directed against surface antigens of this organism. No sample preparation is required. Either frozen or live cells can be detected and quantified. The specificity and sensitivity are assessed by using phytoplankton culture and field samples spiked with a known amount of cultured A. minutum cells. This LFIA is shown to be highly specific for A. minutum and able to detect reproducibly 10(5)cells/L within 30min. The test is applied to environmental samples already characterized by light microscopy counting. No significant difference is observed between the cell densities obtained by these two methods. This handy super-paramagnetic lateral flow immnunoassay biosensor can greatly assist water quality monitoring programs as well as ecological research.

Phytochip: development of a DNA-microarray for rapid and accurate identification of Pseudo-nitzschia spp and other harmful algal species.

Detection of harmful algal blooms has become a challenging concern because of the direct impacts on public health and economy. The identification of toxic dinoflagellates and diatoms in monitoring programs requires an extensive taxonomic expertise and is time consuming. Advances in molecular biology have allowed the development of new approaches, more rapid, accurate and cost-effective for detecting these microorganisms. In this context, we developed a new DNA microarray (called, Phytochip) for the simultaneous detection of multiple HAB species with a particular emphasis on Pseudo-nitzschia species. Oligonucleotide probes were designed along the rRNA operon. After DNA extraction, the target rDNA genes were amplified and labeled using an asymmetric PCR; then, the amplicons were hybridized to the oligonucleotide probes present on the chips. The total assay from seawater sampling to data acquisition can be performed within a working day. Specificity and sensitivity were assessed by using monoclonal cultures, mixtures of species and field samples spiked with a known amount of cultured cells. The Phytochip with its 81 validated oligonucleotide probes was able to detect 12 species of Pseudo-nitzschia and 11 species of dinoflagellates among which were 3 species of Karenia and 3 species of Alexandrium. The Phytochip was applied to environmental samples already characterized by light microscopy and cloned into DNA libraries. The hybridizations on the Phytochip were in good agreement with the sequences retrieved from the clone libraries and the microscopic observations. The Phytochip enables a reliable multiplex detection of phytoplankton and can assist a water quality monitoring program as well as more general ecological research.

Direct and fast detection of Alexandrium minutum algae by using high frequency microbalance.

In this paper, a simple detection of a toxic algae, Alexandrium minutum, was developed using highly sensitive quartz crystal microbalance. In terms of performance, compared with other conventional analytical tools, the main interest of our immunosensor is based on a fast and direct detection of these living cells. This system requires the use of one monoclonal antibody directed against the surface antigen of A. minutum. We demonstrate that the whole living and motile algae are caught and detected. The high specificity of the biosensor is also demonstrated by testing several other dinoflagellate species. The frequency shift is correlated to the A. minutum cell concentration. This simple system is potentially promising for environmental monitoring purposes.

Smelly feet are not always a bad thing: the relationship between cyprid footprint protein and the barnacle settlement pheromone.

A critical phase in the life cycle of sessile benthic marine invertebrates is locating a suitable substratum for settlement. For barnacles, it is the lecithotrophic cypris larva that makes this plankto-benthic transition. In exploring possible substrata for settlement, the cyprid leaves behind 'footprints' of a proteinaceous secretion that reportedly functions as a temporary adhesive, and also acts as a secondary cue in larval-larval interactions at settlement. Here, we show that two polyclonal antibodies raised against peptides localized at the N- and C-terminal regions of the adult settlement cue--the settlement-inducing protein complex (SIPC)--could both detect 'temporary adhesive' indicating that the SIPC is either a component of this secretion or that they are the same protein.

An alpha2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite.

Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the alpha(2)-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral alpha(2)-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions.

Control of flatfish sperm motility by CO2 and carbonic anhydrase.

Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25-100 mM HCO3-, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3-. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3(-)-arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/HCO3(-)-dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm.