Bacterial lipopolysaccharide induces settlement and metamorphosis in a marine larva.

How larvae of the many phyla of marine invertebrates find places appropriate for settlement, metamorphosis, growth, and reproduction is an enduring question in marine science. Biofilm-induced metamorphosis has been observed in marine invertebrate larvae from nearly every major marine phylum. Despite the widespread nature of this phenomenon, the mechanism of induction remains poorly understood. The serpulid polychaete Hydroides elegans is a well established model for investigating bacteria-induced larval development. A broad range of biofilm bacterial species elicit larval metamorphosis in H. elegans via at least two mechanisms, including outer membrane vesicles (OMVs) and complexes of phage-tail bacteriocins. We investigated the interaction between larvae of H. elegans and the inductive bacterium Cellulophaga lytica, which produces an abundance of OMVs but not phage-tail bacteriocins. We asked whether the OMVs of C. lytica induce larval settlement due to cell membrane components or through delivery of specific cargo. Employing a biochemical structure­function approach with a strong ecological focus, the cells and OMVs produced by C. lytica were interrogated to determine the class of the inductive compounds. Here, we report that larvae of H. elegans are induced to metamorphose by lipopolysaccharide produced by C. lytica. The widespread prevalence of lipopolysaccharide and its associated taxonomic and structural variability suggest it may be a broadly employed cue for bacterially induced larval settlement of marine invertebrates.

Laser ablation of the apical sensory organ of Hydroides elegans (Polychaeta) does not inhibit detection of metamorphic cues.

Larvae of many marine invertebrates bear an anteriorly positioned apical sensory organ (ASO) presumed to be the receptor for settlement- and metamorphosis-inducing environmental cues, based on its structure, position and observed larval behavior. Larvae of the polychaete Hydroides elegans are induced to settle by bacterial biofilms, which they explore with their ASO and surrounding anteroventral surfaces. A micro-laser was utilized to destroy the ASO and other anterior ciliary structures in competent larvae of H. elegans. After ablation, larvae were challenged with bacterial biofilmed or clean surfaces and percentage metamorphosis was determined. Ablated larvae were also assessed for cellular damage by applying fluorescently tagged FMRF-amide antibodies and observing the larvae by laser-scanning confocal microscopy. While the laser pulses caused extensive damage to the ASO and surrounding cells, they did not inhibit metamorphosis. We conclude that the ASO is not a required receptor site for cues that induce metamorphosis.

Bacterial envelope polysaccharide cues settlement and metamorphosis in the biofouling tubeworm Hydroides elegans.

Metamorphosis for many marine invertebrates is triggered by external cues, commonly produced by bacteria. For larvae of Hydroides elegans, lipopolysaccharide (LPS) from the biofilm-dwelling bacterium Cellulophaga lytica induces metamorphosis. To determine whether bacterial LPS is a common metamorphosis-inducing factor for this species, we compare larval responses to LPS from 3 additional inductive Gram-negative marine biofilm bacteria with commercially available LPS from 3 bacteria not known to induce metamorphosis. LPS from all the inductive bacteria trigger metamorphosis, while LPS from non-inductive isolated marine bacteria do not. We then ask, which part of the LPS is the inductive element, the lipid (Lipid-A) or the polysaccharide (O-antigen), and find it is the latter for all four inductive bacteria. Finally, we examine the LPS subunits from two strains of the same bacterial species, one inductive and the other not, and find the LPS and O-antigen to be inductive from only the inductive bacterial strain.

The natural sequence of events in larval settlement and metamorphosis of Hydroides elegans (Polychaeta; Serpulidae).

The broadly distributed serpulid worm Hydroides elegans has become a model organism for studies of marine biofouling, development and the processes of larval settlement and metamorphosis induced by surface microbial films. Contrasting descriptions of the initial events of these recruitment processes, whether settlement is induced by (1) natural multi-species biofilms, (2) biofilms composed of single bacterial species known to induce settlement, or (3) a bacterial extract stimulated the research described here. We found that settlement induced by natural biofilms or biofilms formed by the bacterium Pseudoalteromonas luteoviolacea is invariably initiated by attachment and secretion of an adherent and larva-enveloping primary tube, followed by loss of motile cilia and ciliated cells and morphogenesis. The bacterial extract containing complex tailocin arrays derived from an assemblage of phage genes incorporated into the bacterial genome appears to induce settlement events by destruction of larval cilia and ciliated cells, followed by attachment and primary-tube formation. Similar destruction occurred when precompetent larvae of H. elegans or larvae of a nudibranch gastropod were exposed to the extract, although neither of them metamorphosed. We argue that larvae that lose their cilia before attachment would be swept away from the sites that stimulated settlement by the turbulent flow characteristic of most marine habitats.

Formation and Function of the Primary Tube During Settlement and Metamorphosis of the Marine Polychaete Hydroides elegans (Haswell, 1883) (Serpulidae).

AbstractThe serpulid polychaete Hydroides elegans has emerged as a major model organism for studies of marine invertebrate settlement and metamorphosis and for processes involved in marine biofouling. Rapid secretion of an enveloping, membranous, organic primary tube provides settling larvae of H. elegans firm adhesion to a surface and a refuge within which to complete metamorphosis. While this tube is never calcified, it forms the template from which the calcified tube is produced at its anterior end. Examination of scanning and transmission electron micrographs of competent and settling larvae revealed that the tube is secreted from epidermal cells of the three primary segments, with material possibly transported through the larval cuticle via abundant microvilli. The tube is composed of complexly layered fibrous material that has an abundance of the amino acids that characterize the collagenous cuticle of other polychaetes, plus associated carbohydrates. The significance of the dependence on surface bacterial biofilms for stimulating settlement in this species is revealed as a complex interaction between primary tube material, as it is secreted, and the extracellular polymeric substances abundantly produced by biofilm-residing bacteria. This association appears to provide the settling larvae with an adhesion strength similar to that of bacteria in a biofilm and significantly less when larvae settle on a clean surface.

Effects of initial surface wettability on biofilm formation and subsequent settlement of Hydroides elegans.

Hydroides elegans is a major fouling organism in tropical waters around the world, including Pearl Harbor, Hawaii. To determine the importance of initial surface characteristics on biofilm community composition and subsequent colonization by larvae of H. elegans, the settlement and recruitment of larvae to biofilmed surfaces with six different initial surface wettabilities were tested in Pearl Harbor. Biofilm community composition, as determined by a combined approach of denaturing gradient gel electrophoresis and fluorescence in situ hybridization, was similar across all surfaces, regardless of initial wettability, and all surfaces had distinct temporal shifts in community structure over a 10 day period. Larvae settled and recruited in higher numbers to surfaces with medium to low wettability in both May and August, and also to slides with high wettability in August. Pearl Harbor biofilm communities developed similarly on a range of surface wettabilities, and after 10 days in Pearl Harbor all surfaces were equally attractive to larvae of Hydroides elegans, regardless of initial surface properties.

Antifouling character of 'active' hybrid xerogel coatings with sequestered catalysts for the activation of hydrogen peroxide.

Halide-permeable xerogel films prepared from sols containing 50 mol% aminopropyltriethoxysilane (APTES)/50 mol% tetraethoxysilane (TEOS) or 10 mol% APTES/90 mol% TEOS and 0.015 M selenoxide or telluride catalyst in the sol gave reduced settlement of cypris larvae of the barnacle Balanus amphitrite and larvae of the tubeworm Hydroides elegans in the presence of artificial seawater (ASW) and hydrogen peroxide (5-100 microM) relative to glass controls. Settlement of Ulva zoospores was lower on both the 50 mol% APTES/50 mol% TEOS and 10 mol% APTES/90 mol% TEOS xerogel formulations in comparison with glass controls with or without the added catalyst. The 50 mol% APTES/50 mol%TEOS xerogel containing telluride catalyst gave reduced settlement of Ulva zoospores in the presence of 100 microM H(2)O(2) in ASW compared with the same coating without added peroxide. Scanning electron microscopy and XPS data suggest that exposure to H(2)O(2) does not lead to chemical or morphological changes on the xerogel surface.

Complete Genome Sequence of Thalassotalea euphylliae Strain H2.

A bacterial isolate of Thalassotalea euphylliae H2 was collected from the coral Montipora capitata. MinION long reads were employed for scaffolding and complemented with short-read MiSeq sequences to permit complete genome assembly. The genome is approximately 4.36 Mb long, with 3,669 protein-coding genes, 92 tRNAs, and 21 rRNAs.

Microbial biofilms facilitate adhesion in biofouling invertebrates.

Much interest has focused on the role of microbial layers--biofilms--in stimulating attachment of invertebrates and algae to submerged marine surfaces. We investigated the influence of biofilms on the adhesion strength of settling invertebrates. Larvae of four species of biofouling invertebrate were allowed to attach to test surfaces that were either clean or coated with a natural biofilm. Measuring larval removal under precisely controlled flow forces, we found that biofilms significantly increased adhesion strength in the ascidian Phallusia nigra, the polychaete tubeworm Hydroides elegans, and the barnacle Balanus amphitrite at one or more developmental stages. Attachment strength in a fourth species, the bryozoan Bugula neritina, was neither facilitated nor inhibited by the presence of a biofilm. These results suggest that adhesive strength and perhaps composition may vary across different invertebrate taxa at various recruitment stages, and mark a new path of inquiry for biofouling research.

Full-Genome Sequence of Thalassotalea euphylliae H1, Isolated from a Montipora capitata Coral Located in Hawai'i.

The isolate of Thalassotalea euphylliae H1 was collected from the surface of a Montipora capitata coral. The genome was assembled using long reads from a Nanopore MinION sequencer for scaffolding and complemented with short-read MiSeq sequences. The genome was approximately 4.77 Mb long with 4,020 protein-coding genes, 92 tRNAs, and 22 rRNAs.

Induction of Invertebrate Larval Settlement; Different Bacteria, Different Mechanisms?

Recruitment via settlement of pelagic larvae is critical for the persistence of benthic marine populations. For many benthic invertebrates, larval settlement occurs in response to surface microbial films. Larvae of the serpulid polychaete Hydroides elegans can be induced to settle by single bacterial species. Until now, only Pseudoalteromonas luteoviolacea had been subjected to detailed genetic and mechanistic studies. To determine if the complex structures, termed tailocins, derived from phage-tail gene assemblies and hypothesized to be the settlement cue in P. luteoviolacea were present in all inductive bacteria, genomic comparisons with inductive strains of Cellulophaga lytica, Bacillus aquimaris and Staphylococcus warneri were undertaken. They revealed that the gene assemblies for tailocins are lacking in these other bacteria. Negatively stained TEM images confirmed the absence of tailocins and revealed instead large numbers of extracellular vesicles in settlement-inductive fractions from all three bacteria. TEM imaging confirmed for C. lytica that the vesicles are budded from cell surfaces in a manner consistent with the production of outer membrane vesicles. Finding multiple bacteria settlement cues highlights the importance of further studies into the role of bacterial extracellular vesicles in eliciting settlement and metamorphosis of benthic marine larvae.

Expression and Localization of Carbonic Anhydrase Genes in the Serpulid Polychaete Hydroides elegans.

The metalloenzyme, carbonic anhydrase (CA), catalyzes the reversible hydration of carbon dioxide into bicarbonate, and is responsible for biomineralization processes in animals. In the Annelida, the marine worms in the family Serpulidae are typified by the construction of calcium carbonate tubes. Hydroides elegans, a common member of warmwater biofouling communities around the world, provides an outstanding model for studies of calcification. To better understand the molecular process of biomineralization in H. elegans, we searched transcriptomes for CA genes at several life-history stages. Twelve CA genes were recovered in the transcriptomes. Whole mount in situ hybridization was performed for two of those genes on larvae and calcifying juveniles. A cytosolic CA isoform, HeCA1, and a secreted CA isoform, HeCA2, were expressed within the collar segment corresponding to the location of glands involved in formation of the calcified tube. Expression of these genes within collar segment tissues supports the role of CAs in generating bicarbonate for biomineralization processes. A phylogenetic tree of the α-CA gene family was constructed to increase understanding of CA-gene evolution within the family and its relationship to CA genes among the Metazoa.

Fouling-Release Performance of Silicone Oil-Modified Siloxane-Polyurethane Coatings.

The effect of incorporation of silicone oils into a siloxane-polyurethane fouling-release coatings system was explored. Incorporation of phenylmethyl silicone oil has been shown to improve the fouling-release performance of silicone-based fouling-release coatings through increased interfacial slippage. The extent of improvement is highly dependent upon the type and composition of silicone oil used. The siloxane-polyurethane (SiPU) coating system is a tough fouling-release solution, which combines the mechanical durability of polyurethane while maintaining comparable fouling-release performance with regard to commercial standards. To further improve the fouling-release performance of the siloxane-PU coating system, the use of phenylmethyl silicones oils was studied. Coatings formulations were prepared incorporating phenylmethyl silicone oils having a range of compositions and viscosities. Contact angle and surface energy measurements were conducted to evaluate the surface wettability of the coatings. X-ray photoelectron spectroscopy (XPS) depth profiling experiments demonstrated self-stratification of silicone oil along with siloxane to the coating-air interface. Several coating formulations displayed improved or comparable fouling-release performance to commercial standards during laboratory biological assay tests for microalgae (Navicula incerta), macroalgae (Ulva linza), adult barnacles (Balanus amphitrite syn. Amphibalanus amphitrite), and mussels (Geukensia demissa). Selected silicone-oil-modified siloxane-PU coatings also demonstrated comparable fouling-release performance in field immersion trials. In general, modifying the siloxane-PU fouling-release coatings with a small amount (1-5 wt % basis) of phenylmethyl silicone oil resulted in improved performance in several laboratory biological assays and in long-term field immersion assessments.

Contact angle anomalies indicate that surface-active eluates from silicone coatings inhibit the adhesive mechanisms of fouling organisms.

Silicone coatings with critical surface tensions (CST) between 20 and 30 mN m-1 more easily release diverse types of biofouling than do materials of higher and lower CST. Oils added to these coatings selectively further diminish the attachment strengths of different marine fouling organisms, without significantly modifying the initial CST. In a search for the mechanisms of this improved biofouling resistance, the interfacial instabilities of four silicone coatings were characterised by comprehensive contact angle analyses, using up to 12 different diagnostic fluids selected to mimic the side chain chemistries of the common amino acids of bioadhesive proteins. The surfaces of painted steel test panels were characterised both before and after exposure to freshwater, brackish water, and seawater over periods ranging from 9 months to nearly 4 years. Contact angle measurements demonstrated significant surface activity of the oil-amended coatings both before and after long-term underwater exposure. The surface activity of the control (coating without oil) increased as a result of underwater exposure, consistent with mild surface chain scission and hydrolysis imparting a self-surfactancy to the coating and providing a weak boundary layer promoting continuing easy release of attaching foulants. Coatings with additives that most effectively reduced biofouling showed both initial and persistent contact angle anomalies for the test liquid, thiodiglycol, suggesting lower-shear biofouling release mechanisms based upon diminished bioadhesive crosslinking by interfering with hydrogen- and sulfhydryl bonds. Swelling of the silicone elastomeric coatings by hydrocarbon fluids was observed for all four coatings, before and after immersion.

Interspecific variation in patterns of adhesion of marine fouling to silicone surfaces.

The adhesion of six fouling organisms: the barnacle Balanus eburneus, the gastropod mollusc Crepidulafornicata, the bivalve molluscs Crassostrea virginica and Ostrea/Dendrostrea spp., and the serpulid tubeworms Hydroides dianthus and H. elegans, to 12 silicone fouling-release surfaces was examined. Removal stress (adhesion strength) varied among the fouling species and among the surfaces. Principal component analysis of the removal stress data revealed that the fouling species fell into two distinct groups, one comprising the bivalve molluscs and tubeworms, and the other the barnacle and the gastropod mollusc. None of the silicone materials generated a minimum in removal stress for all the organisms tested, although several surfaces produced low adhesion strengths for both groups of species. These results suggest that fouling-release materials do not rank (in terms of adhesion strength) identically for all fouling organisms, and thus development of a globally-effective hull coating will continue to require testing against a diversity of encrusting species.