Settlement behavior of zoospores of Ulva linza during surface selection studied by digital holographic microscopy.

Settlement of the planktonic dispersal stages of marine organisms is the crucial step for the development of marine biofouling. Four-dimensional holographic tracking reveals the mechanism by which algal spores select surfaces suitable for colonization. Quantitative analysis of the three dimensional swimming trajectories of motile spores of a macroalga (Ulva linza) in the vicinity of surfaces functionalized with different chemistries reveals that their search strategy and swimming behavior is correlated to the number of settled spores found in spore settlement bioassays conducted over 45 min. The spore motility and exploration behavior can be classified into different motion patterns, with their relative occurrence changing with the surface chemistry. Based on the detailed motility analysis we derived a model for the surface selection and settlement process of Ulva zoospores.

Digital in-line X-ray holography with zone plates.

Single pulse imaging with radiation provided by free-electron laser sources is a promising approach towards X-ray microscopy, which is expected to provide high resolution images of biological samples unaffected by radiation damage. One fully coherent imaging technique for this purpose is digital in-line holography. Key to its successful application is the creation of X-ray point sources with high photon flux. In this study we applied zone plates to create such point sources with synchrotron radiation provided by the storage ring BESSY II. The obtained, divergent light cone is applied to holographic microscopy of biological objects such as critical point dried Navicula perminuta diatoms and human cells using photons with an energy of 250 eV. Compared to conventional experiments employing pinholes, exposure times are reduced by two orders of magnitude.

X-ray holographic microscopy with zone plates applied to biological samples in the water window using 3rd harmonic radiation from the free-electron laser FLASH.

The imaging of hydrated biological samples - especially in the energy window of 284-540 eV, where water does not obscure the signal of soft organic matter and biologically relevant elements - is of tremendous interest for life sciences. Free-electron lasers can provide highly intense and coherent pulses, which allow single pulse imaging to overcome resolution limits set by radiation damage. One current challenge is to match both the desired energy and the intensity of the light source. We present the first images of dehydrated biological material acquired with 3rd harmonic radiation from FLASH by digital in-line zone plate holography as one step towards the vision of imaging hydrated biological material with photons in the water window. We also demonstrate the first application of ultrathin molecular sheets as suitable substrates for future free-electron laser experiments with biological samples in the form of a rat fibroblast cell and marine biofouling bacteria Cobetia marina.

Interactions of zoospores of Ulva linza with arginine-rich oligopeptide monolayers.

We recently reported on the strong interactions of zoospores of the green alga, Ulva linza with an arginine-rich oligopeptide self-assembled monolayer (SAM) [Biofouling 2008, 24, 303-312], where the arginine-rich peptide induced not only high spore settlement, but also a form of abnormal settlement, or "pseudo-settlement", whereby a proportion of spores do not go through the normal process of surface exploration, adhesive exocytosis, and loss of flagella. Further, it was demonstrated that both the total number of settled spores and the fraction of pseudosettled spores were related to the surface density of the arginine-rich peptide. Here we present a further investigation of the interactions of zoospores of Ulva with a set of oligomeric, de novo designed, arginine-rich peptides, specifically aimed to test the effect of peptide primary structure on the interaction. Via variations in the peptide length and by permutations in the amino acid sequences, we gain further insight into the spore-surface interactions. The interpretation of the biological assays is supported by physicochemical characterization of the SAMs using infrared spectroscopy, ellipsometry, and contact angle measurements. Results confirm the importance of arginine residues for the anomalous pseudosettlement, and we found that settlement is modulated by variations in both the total length and peptide primary structure. To elucidate the causes of the anomalous settlement and the possible relation to peptide-membrane interactions, we also compared the settlement of the "naked" zoospores of Ulva (which present a lipoprotein membrane to the exterior without a discrete polysaccharide cell wall), with the settlement of diatoms (unicellular algae that are surrounded by a silica cell wall), onto the peptide SAMs. Cationic SAMs do not notably affect settlement (attachment), adhesion strength, or viability of diatom cells, suggesting that the effect of the peptides on zoospores of Ulva is mediated via specific peptide-membrane interactions.

Anomalous settlement behavior of Ulva linza zoospores on cationic oligopeptide surfaces.

Identification of settlement cues for marine fouling organisms opens up new strategies and methods for biofouling prevention, and enables the development of more effective antifouling materials. To this end, the settlement behaviour of zoospores of the green alga Ulva linza onto cationic oligopeptide self-assembled monolayers (SAMs) has been investigated. The spores interact strongly with lysine- and arginine-rich SAMs, and their settlement appears to be stimulated by these surfaces. Of particular interest is an arginine-rich oligopeptide, which is effective in attracting spores to the surface, but in a way which leaves a large fraction of the settled spores attached to the surface in an anomalous fashion. These 'pseudo-settled' spores are relatively easily detached from the surface and do not undergo the full range of cellular responses associated with normal commitment to settlement. This is a hitherto undocumented mode of settlement, and surface dilution of the arginine-rich peptide with a neutral triglycine peptide demonstrates that both normal and anomalous settlement is proportional to the surface density of the arginine-rich peptide. The settlement experiments are complemented with physical studies of the oligopeptide SAMs, before and after extended immersion in artificial seawater, using infrared spectroscopy, null ellipsometry and contact angle measurements.

The influence of surface lubricity on the adhesion of Navicula perminuta and Ulva linza to alkanethiol self-assembled monolayers.

The settlement and adhesion of Navicula perminuta and Ulva linza to methyl-terminated alkanethiol self-assembled monolayers (SAMs) of increasing chain length has been investigated. Organisms were allowed to settle onto the monolayers and were subsequently exposed to hydrodynamic shear stress in order to determine their adhesion strength. Results show that as the SAM structure changes from amorphous to crystalline (C14), there is a marked change in the adhesion of N. perminuta and U. linza. Given that the SAMs in the series all exhibit similar contact angle behaviour and surface energy, it is hypothesized that the lubricity of the surface plays a role in determining the surface adhesion.

Activity of commercial enzymes on settlement and adhesion of cypris larvae of the barnacle Balanus amphitrite, spores of the green alga Ulva linza, and the diatom Navicula perminuta.

Fouling species produce adhesive polymers during the settlement, adhesion and colonization of new surfaces in the marine environment. The present paper tests the hypothesis that enzymes of the appropriate specificity may prevent biofouling by hydrolysing these adhesive polymers. Seventeen commercially available enzyme preparations designed originally for bulk use in a range of end-use applications were tested for their effects on the settlement and/or adhesion of three major fouling species, viz. the green alga Ulva linza, the diatom Navicula perminuta and the barnacle Balanus amphitrite. The serine-proteases were found to have the broadest antifouling potential reducing the adhesion strength of spores and sporelings of U. linza, cells of N. perminuta and inhibiting settlement of cypris larvae of B. amphitrite. Mode-of-action studies on the serine-protease, Alcalase, indicated that this enzyme reduced adhesion of U. linza in a concentration-dependent manner, that spores of the species could recover their adhesive strength if the enzyme was removed and that the adhesive of U. linza and juvenile cement of B. amphitrite became progressively less sensitive to hydrolysis as they cured.