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Barnacle shell (BSh)-associated bacteria, their culture supernatants (CS), and biofilm exopolymers (BE) are known to induce cyprid metamorphosis of Balanus amphitrite. Besides bacteria, the BSh also harbors diatoms. However, the roles of their CS and BE on cyprid metamorphosis were not known, and the same was evaluated using two epibiotic diatoms, Achnanthes longipes and Melosira nummuloides, isolated from the BSh. The CS of M. nummuloides, a waterborne cue, promoted cyprid metamorphosis only when isolated in the presence of BSh, whereas that of A. longipes inhibited the same. The high-performance liquid chromatography (HPLC) of CS of both the diatoms indicated the dominance of arabinose, an inhibitory sugar. However, thiols were additionally present in the CS of M. nummuloides as indicated by fourier-transform infrared spectroscopy (FTIR) and are probably responsible for their promotory effect. The BE, a surface-bound cue, of both the diatoms facilitated metamorphosis only when isolated in the presence of BSh and were rich in N-acetyl-D-glucosamine (NAG). Thus, the interaction of NAG with the shell proteins probably provides a set of cues that facilitate cyprid metamorphosis of B. amphitrite. [ABSTRACT FROM AUTHOR]

Nitric Oxide (NO) signaling is known to govern the cyprid metamorphosis of barnacle Balanus amphitrite , a potential biofouling organism. This signaling cascade is mainly driven by two important enzymes, Nitric Oxide Synthase (NOS) and cyclic Guanosine monophosphate (cGMP)-specific Phosphodiesterase (PDE), which exist in multiple isoforms. But, involvement of the exact isoform of these enzymes was unknown, and the same was addressed in the present study using pharmacological compounds, targeting the specific isoforms of NOS and PDE. When the cyprids were treated with specific NOS inhibitors (7-Nitroindazole for NOS I and S-methylisothiourea hemisulphate for NOS II), the metamorphosis was promoted indicating the involvement of both NOS I and NOS II. However, when cyprids were exposed to PDE inhibitors (Sildenafil, Dipyridamole and Bay 73–6691 targeting PDE5, PDE6 and PDE9 respectively), metamorphosis was significantly inhibited only in case of Sildenafil, indicating the involvement of PDE5. The expression of PDE5 in the cyprid was demonstrated using immunofluorescence technique. Further, it was also demonstrated that the interplay between NO and Serotonin signaling plays a vital role in the pelago-benthic transition of B. amphitrite . Targeting these signaling pathways in the future could provide an antifouling solution against barnacles. [ABSTRACT FROM AUTHOR]

In this study, as part of our continuous search for environmentally-friendly antifoulants from natural resources, subergorgic acid (SA) was identified from the gorgonian coral Subergorgia suberosa, demonstrating non-toxic, significant inhibitory effects (EC50 1.25 μg/mL, LC50 > 25 μg/mL) against the settlement of Balanus amphitrite. To further explore the bioactive functional groups of SA and synthesize more potent antifouling compounds based on the lead SA, the structure-activity relationships of SA were studied, followed by rational design and synthesis of two series of SA derivatives (one being benzyl esters of SA and another being SA derivatives containing methylene chains of various lengths). Our results indicated that (1) both the double bond and ketone carbonyl are essential elements responsible for the antifouling effect of SA, while the acid group is not absolutely necessary for maintaining the antifouling effect; (2) all benzyl esters of SA displayed good antifouling effects (EC50 ranged from 0.30 to 2.50 μg/mL) with the most potent compound being 5 (EC50 0.30 μg/mL, LC50 > 25 μg/mL), which was over four-fold more potent than SA; and (3) the introduction of a methylene chain into SA reduces the antifouling potency while the length of the methylene chain may differently influence the antifouling effect, depending on the functional group at the opposite site of the methylene chain. Not only has this study successfully revealed the bioactive functional groups of SA, contributing to the mechanism of SA against the settlement of B. amphitrite, but it has also resulted in the identification of a more potent compound 5, which might represent a non-toxic, high-efficiency antifoulant.

A series of 30 sclerotioramine derivatives ( 2- 31) of the natural compound, (+)-sclerotiorin ( 1), has been successfully semi-synthesized by a one-step reaction with high yields (up to 80%). The structures of these new derivatives were established by extensive spectroscopic methods and single-crystal X-ray diffraction analysis for 3, 6, and 10. (+)-Sclerotiorin ( 1) and its semisynthetic derivatives ( 2- 31) were evaluated for their antifouling activity. Most of them except 6, 7, 8, 12, and 28 showed potent antifouling activity against the larval settlement of the barnacle Balanus amphitrite. More interestingly, most of the aromatic amino-derivatives ( 13- 17, 19- 21, 23, 25- 27, and 29- 31) showed strong antifouling activity; however, only two aliphatic amino-derivatives ( 5 and 10) had the activity. [ABSTRACT FROM AUTHOR]

Surface-active amphiphilic diblock copolymers, Si-EFS14 and Si-EFS71, consisting of a poly(dimethyl siloxane) block (degree of polymerisation 11) and a poly(4-(triethyleneglycol monomethyl ether)-2,3,5,6-tetrafluorostyrene) block (average degree of polymerisation 14 and 71) were synthesised by ATRP. Films were prepared by incorporating each copolymer (4 wt%) into a PDMS matrix, which was then condensation cured. Bioassays were performed on the films using two barnacle species, Balanus amphitrite and Balanus improvisus , at different stages of their life cycles. The cyprids of B. improvisus settled on all test surfaces in higher numbers than those of B. amphitrite . However, the juveniles of B. improvisus were more easily removed from the films containing the copolymer Si-EFS14 than from those containing the copolymer Si-EFS71. An XPS analysis revealed that the near-surface region of copolymer Si-EFS71 was enriched in oxyethylenic chains and became even more populated by these hydrophilic chains after the films were immersed in water. [ABSTRACT FROM AUTHOR]

Marine organism-derived secondary metabolites are promising potential sources for discovering environmentally safe antifouling agents. In present study, 55 marine secondary metabolites and their synthesized derivatives were tested and evaluated for their antifouling activities and security. These compounds include 44 natural products isolated from marine invertebrates and their symbiotic microorganisms collected from the South China Sea and 11 structural modified products derived from the isolated compounds. The natural secondary metabolites, covering phenyl ether derivatives, terpenoids, 9, 11-secosteroids, anthraquinones, alkaloids, nucleoside derivatives and peptides, were isolated from two corals, one sponge and five symbiotic fungi. All of the isolated and synthesized compounds were tested for their antifouling activities against the cyprids of barnacle Balanus ( Amphibalanus) amphitrite Darwin. Noticeably, five phenyl ether derivatives ( 9, 11, 13- 15) exhibited potent anti-larval settlement activity with the EC values lower than 3.05 μM and the LC/EC ratios higher than 15. The study of structure-activity relationship (SAR) revealed that the introduction of acetoxy groups and bromine atoms to phenyl ether derivatives could significantly improve their antifouling activities. This is the first report on the SAR of phenyl ether derivatives on antifouling activity against barnacle B. amphitrite. The polybrominated diphenyl ether derivative, 2, 4, 6, 2′, 4′, 6′-hexabromo-diorcinol ( 13), which displayed excellent antifouling activity, was considered as a promising candidate of environmentally friendly antifouling agents. [ABSTRACT FROM AUTHOR]

Both the whole cells and protoplasts of Pseudomonas aeruginosa PAO1 and Bacillus cereus , two biofilm-forming bacteria, were disrupted by the lipopeptide 6-2 produced by Bacillus amyloliquefaciens anti-CA. The lipopeptide 6-2 could also effectively inhibit the formation of biofilms and disperse pre-formed biofilms. Live/dead staining of the biofilms grown in the absence or presence of the lipopeptide 6-2 showed that more dead bacterial cells in the presence of the lipopeptide than those in the absence of the lipopeptide and biofilm formation was greatly reduced by the lipopeptide 6-2. Expression of the PslC gene related to exopolysaccharides in P. aeruginosa PAO1 was also inhibited. All these results demonstrated that the lipopeptide 6-2 produced by B. amyloliquefaciens anti-CA had a high activity against biofilm-forming bacteria. The lipopeptide 6-2 also killed the larvae of Balanus amphitrite and inhibit the germination of Laminaria japonica spore and growth of protozoa, all of which were the fouling organisms in marine environments. [ABSTRACT FROM AUTHOR]

Three species of sponges and a tunicate were collected from Obhur creek of Jeddah coast for this bioactivity study. In order to assess the antifouling efficacy of selected marine organisms, methanolic extracts of these organisms were tested against different fouling bacterial forms and II-instar stage of the barnacle, Balanus amphitrite. Antibiosis, bioactivity and followed by multivariate analyses were carried out to check the efficacy of antifouling effect of the selected marine organisms. Principal component analysis revealed the exemplary antifouling efficacy of the sponge extracts of Stylissa sp. observed followed by Hyrtios sp. against bacterial forms in the laboratory study. De-trended correspondence analysis confirmed that the contribution of antifouling efficacy of the selected sponge extracts was observed to be more towards Bacillus sp., Vibrio sp. and Alteromonas sp. Moreover, the efficacy of Hyrtios sp. extract (20.430 μg mL) followed by Stylissa sp. (30.945 μg mL) showed higher against barnacle instar compared with other extracts in the bioactivity assay. Bray-Curtis cluster analysis under paired linkage categorized all the sponge extracts into one major cluster with 75% similarity, and one outlier tunicate. More than 80% similarity observed between Hyrtios sp. and Stylissa sp. Fourier transform infrared spectroscopy (FTIR) showed that the contribution of major peaks found in the marine organisms were towards sulfones, sulfoxides, cyanates and ketones. [ABSTRACT FROM AUTHOR]

"Ocean acidification" (OA), a change in seawater chemistry driven by increased uptake of atmospheric CO2 by the oceans, has probably been the most-studied single topic in marine science in recent times. The majority of the literature on OA report negative effects of CO2 on organisms and conclude that OA will be detrimental to marine ecosystems. As is true across all of science, studies that report no effect of OA are typically more difficult to publish. Further, the mechanisms underlying the biological and ecological effects of OA have received little attention in most organismal groups, and some of the key mechanisms (e.g. calcification) are still incompletely understood. For these reasons, the ICES Journal of Marine Science solicited contributions to this special issue. In this introduction, I present a brief overview of the history of research on OA, call for a heightened level of organized (academic) scepticism to be applied to the body of work on OA, and briefly present the 44 contributions that appear in this theme issue. OA research has clearly matured, and is continuing to do so. We hope that our readership will find that, when taken together, the articles that appear herein do indeed move us "Towards a broader perspective on ocean acidification research". [ABSTRACT FROM AUTHOR]

Ocean acidification (OA) is anticipated to interact with the more frequently occurring hypoxic conditions in shallow coastal environments. These could exert extreme stress on the barnacle-dominated fouling communities. However, the interactive effect of these two emerging stressors on early-life stages of fouling organisms remains poorly studied. We investigated both the independent and interactive effect of low pH (7.6 vs. ambient 8.2) and low oxygen (LO; 3 mg l-1 vs. ambient 5 mg l-1) from larval development through settlement (attachment and metamorphosis) and juvenile growth of the widespread fouling barnacle, Balanus amphitrite. In particular, we focused on the critical transition between planktonic and benthic phases to examine potential limiting factors (i.e. larval energy storage and the ability to perceive cues) that may restrain barnacle recruitment under the interactive stressors. LO significantly slowed naupliar development, while the interaction with low pH (LO-LP) seemed to alleviate the negative effect. However, 20-50% of the larvae became cyprid within 4 d post-hatching, regardless of treatment. Under the two stressors interaction (LO-LP), the barnacle larvae increased their feeding rate, which may explain why their energy reserves at competency were not different from any other treatment. In the absence of a settlement-inducing cue, a significantly lower percentage of cyprids (~15% lower) settled in LO and LO-LP. The presence of an inducing cue, however, elevated attachment up to 50-70% equally across all treatments. Post-metamorphic growth was not altered, although the condition index was different between LO and LO-LP treatments, potentially indicating that less and/or weaker calcified structures were developed when the two stressors were experienced simultaneously. LO was the major driver for the responses observed and its interaction with low pH should be considered in future studies to avoid underestimating the sensitivity of biofouling species to OA and associated climate change stressors. [ABSTRACT FROM AUTHOR]

A new symmetric 16-membered macrodiolide, trichobotryside A ( 1 ), and two new asymmetric 18-membered macrodiolides trichobotrysides B–C ( 2 – 3 ) were obtained from the deep-sea-derived fungus Trichobotrys effuse DFFSCS021. Their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of 1 and 2 were established by the methanolysis and modified Mosher method. And the methanolysis of both 1 and 2 produced the new compound of 4 R ,7 S ,9 S -trihydroxyl-2-en-1-methyl decanoate ( 4 ). It was rare to obtain an 18-membered macrodiolide from nature. Compound 1 showed strong antifouling activity against Bugula neritina and Balanus amphitrite larvae settlement. [ABSTRACT FROM AUTHOR]

Barnacle larvae (cyprids) explore surfaces to identify suitable settlement sites. This process is selective, and cyprids respond to numerous surface cues. To better understand the settlement process, it is desirable to simultaneously monitor both the surface exploration behavior and any close interactions with the surface. Stereoscopic 3D tracking of the cyprids provides quantitative access to surface exploration and pre-settlement rituals. Imaging surface plasmon resonance (SPR) reveals any interactions with the surfaces, such as surface inspection during bipedal walking and deposition of temporary adhesives. We report on a combination of both techniques to bring together information on swimming behavior in the vicinity of the interface and physical interactions of the cyprid with the surface. The technical requirements are described, and we applied the setup to cyprids of Balanus amphitrite . Initial data shows the applicability of the combined instrument to correlate exploration and touchdown events on surfaces with different chemical termination. [ABSTRACT FROM AUTHOR]

Block copolymers made from a poly(dimethyl siloxane) (Si) and a poly(meth)acrylate carrying oxyethylene (EG) or fluoroalkyl (AF) side chains were synthesized and incorporated as surface-active components into a silicone matrix to produce cross-linked films with different surface hydrophilicity/phobicity. Near-edge X-ray absorption fine structure (NEXAFS) studies showed that film surfaces containing Si-EG were largely populated by the siloxane, with the oxyethylene chains present only to a minor extent. In contrast, the fluorinated block was selectively segregated to the polymer–air interface in films containing Si-AF as probed by NEXAFS and X-ray photoelectron spectroscopy (XPS) analyses. Such differences in surface composition were reflected in the biological performance of the coatings. While the films with Si-EG showed a higher removal of bothUlva linzasporelings andBalanus amphitritejuveniles than the silicone control, those with Si-AF exhibited excellent antifouling properties, preventing the settlement of cyprids ofB. amphitrite. [ABSTRACT FROM AUTHOR]

Turbinaria ornata mediated silver nanoparticles (TOAg-NPs) were evaluated for antibacterial activity against 15 biofilm forming bacterial isolates. A field study in natural seawater for 60 days showed antifouling activity of TOAg-NPs on stainless steel coupons (SS-304) coated with Apcomin zinc chrome (AZC) primer. Though TOAg-NPs showed broad spectrum of antibacterial activity, the maximum zone of inhibition was with Escherichia coli (71.9%) and a minimum with Micrococcus sp. (40%) due to the EPS secretion from Gram-positive bacteria. Compared to control coupons (18.9 [× 10 3 ], 67.0 [× 10 3 ], 13.5 [× 10 4 ] and 24.7 [× 10 4 ] CFU/cm 2 ), experimental biocide coupons (71.0 [× 10 2 ], 32.0 [× 10 3 ], 82.0 [× 10 3 ] and 11.3 [× 10 4 ] CFU/cm 2 ) displayed lesser bacterial population density. Toxicity studies revealed 100% mortality for Balanus amphitrite larvae at 250 μg ml − 1 concentration within 24 h, while 56.6% recorded for Artemia marina at the same concentration indicating less toxicity to non target species. It proved that AZC + TOAg-NPs prevent biofouling by its Ag-NS affinity and antimicrobial effectivity. [ABSTRACT FROM AUTHOR]

Background: The barnacle Balanus amphitrite is widely distributed in marine shallow and tidal waters, and has significant economic and ecological importance. Nauplii, the first larval stage of most crustaceans, are extremely abundant in the marine zooplankton. However, a lack of genome information has hindered elucidation of the molecular mechanisms of development, settlement and survival strategies in extreme marine environments. We sequenced and constructed the genome dataset for nauplii to obtain comprehensive larval genetic information. We also investigated iTRAQ-based protein expression patterns to reveal the molecular basis of nauplii development, and to gain information on larval survival strategies in the Red Sea marine environment. Results: A nauplii larval transcript dataset, containing 92,117 predicted open reading frames (ORFs), was constructed and used as a reference for the proteome analysis. Genes related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. We observed remarkable plasticity in the proteome of Red Sea larvae. The proteins associated with development, stress responses and osmoregulation showed the most significant differences between the two larval populations studied. The synergistic overexpression of heat shock and osmoregulatory proteins may facilitate larval survival in intertidal habitats or in extreme environments. Conclusions: We presented, for the first time, comprehensive transcriptome and proteome datasets for Red Sea nauplii. The datasets provide a foundation for future investigations focused on the survival mechanisms of other crustaceans in extreme marine environments. [ABSTRACT FROM AUTHOR]

In the present study, we employed the widespread and gregarious barnacle species Balanus amphitrite in a biomonitoring program to evaluate coastal pollution around three piers (i.e., Tso Wo Hang, Sai Kung and Hebe Haven) in Hong Kong. An integrated approach was used herein, combining both the chemical determination of contaminant concentrations, including metals and polybrominated diphenyl ethers (PBDEs), and a suite of biological responses across the entire barnacle lifecycle (i.e., adult, nauplius, cyprid and juvenile). The analytical results revealed a distinct geographical distribution of metals and PBDEs. Adult physiological processes and larval behaviors varied significantly among the three piers. Furthermore, a correlation analysis demonstrated a specific suite of biological responses towards metal and PBDE exposure, likely resulting from their distinct modes of action. Overall, the results of this study indicated that the combination of chemical and biological tests provided an integrated measure for the comprehensive assessment of marine pollution. [ABSTRACT FROM AUTHOR]

The projected increase in the pCO 2 and temperature level by the end of the 21st century, is expected to influence the life cycle of marine invertebrates. Experiments were carried out with an acorn barnacle, Balanus amphitrite a dominant inter-tidal organism, to investigate the effect of these variables on its larval development and metamorphosis. Four alternate combinations of temperature and pCO 2 : (1) control (~ 30 °C and ~ 400 μatm); (2) elevated pCO 2 (~ 30 °C and ~ 750 μatm); (3) elevated temperature (~ 34 °C and ~ 400 μatm); and (4) synergistic (~ 34 °C and ~ 750 μatm) were tested. Apart from their direct effect, the diet-mediated influence was also evaluated by providing a diatom feed ( Chaetoceros calcitrans ) that was grown under similar conditions as that of nauplii. In both the direct and diet-mediated effects, irrespective of pCO 2 levels, elevated temperature favoured faster naupliar development. However, in the direct effect, the synergistic condition yielded poor quality cyprids (non-feeding, pre settlement larval stage) as evidenced by lower RNA:DNA ratio, that resulted in lower metamorphosis. On the contrary, in the diet-mediated evaluation, the feed grown in the synergistic condition had higher protein content that facilitated faster naupliar development, better quality of cyprids (higher RNA:DNA ratio) and yielded a higher percentage of metamorphosis. The results suggest that future climatic conditions will affect the quality of food consumed by invertebrate larvae, thus influencing their development and metamorphosis. Therefore, future studies evaluating the climate change scenarios to depict the changes in settlement and recruitment of marine invertebrates should be advocated to integrate the changes in the food chain. [ABSTRACT FROM AUTHOR]

Effective and ecofriendly antifouling (AF) compounds have been arising from naturally produced chemicals. The objective of this study is to use cyanobacteria-derived agents to investigate the role of acetylcholinesterase (AChE) activity as an effect and/or mode of action of promising AF compounds, since AChE inhibitors were found to inhibit invertebrate larval settlement. To pursue this objective, in vitro quantification of AChE activity under the effect of several cyanobacterial strain extracts as potential AF agents was performed along with in vivo AF (anti-settlement) screening tests. Pre-characterization of different cholinesterases (ChEs) forms present in selected tissues of important biofouling species was performed to confirm the predominance of AChE, and an in vitro AF test using pure AChE activity was developed. Eighteen cyanobacteria strains were tested as source of potential AF and AChE inhibitor agents. Results showed effectiveness in selecting promising eco-friendly AF agents, allowing the understanding of the AF biochemical mode of action induced by different compounds. This study also highlights the potential of cyanobacteria as source of AF agents towards invertebrate macrofouling species. [ABSTRACT FROM AUTHOR]

Cuprous oxide is the most commonly used biocide in antifouling paints. However, copper has harmful effects not only on the fouling community but also on non-target species. In the current study, we investigated the use of thymol, eugenol and guaiacol in this role combined with small quantities of copper. Phenolic compounds were tested for anti-settlement activity against cyprid larvae of the barnacle Balanus amphitrite and for their toxicity to nauplius larvae. Thymol, eugenol and guaiacol were active for anti-settlement but guaiacol had the disadvantage of being toxic to nauplius larvae. However, all of them showed therapeutic ratio>1. Antifouling paints with thymol (low copper content/thymol, LCP/T), eugenol (low copper content/eugenol, LCP/E) and guaiacol (low copper content/guaiacol, LCP/G) combined with small copper content were formulated for field trials. After 12 months exposure in the sea, statistical analysis revealed that LCP/T and LCP/E paints were the most effective combinations and had similar performances to control paints with high copper content (traditional cuprous oxide based paints). In contrast, LCP/G paint was only partially effective in preventing and inhibiting biofouling and was colonized by some hard and soft foulers. However, this antifouling paint was effective against calcareous tubeworm Hydroides elegans . In the light of various potential applications, thymol, eugenol and guaiacol have thus to be considered in future antifouling formulations. [ABSTRACT FROM AUTHOR]

Marine biofouling has a major economic impact, especially when it occurs on ship hulls or aquaculture facilities. Since the International Maritime Organization (IMO) treaty to ban the application of organotin-based paints to ships went into effect in 2008, there is an urgent demand for the development of efficient and environmentally friendly antifouling agents. Marine microorganisms have proved to be a potential source of antifouling natural compounds. In this study, six dihydroquinolin-2-one-containing alkaloids, three monoterpenoids combined with a 4-phenyl-3,4-dihydroquinolin-2(1 H)-one ( 1- 3) and three 4-phenyl-3,4-dihydroquinolin-2(1 H)-one alkaloids ( 4- 6), were isolated from the gorgonian coral-derived fungus Scopulariopsis sp. collected in the South China Sea. These dihydroquinolin-2-one-containing alkaloids were evaluated against the larval settlement of barnacle Balanus amphitrite, and antifouling activity was detected for the first time for this class of metabolites. All of them except 6 showed strong antifouling activity. Compounds 1 and 2 were discovered to be the most promising non-toxic antilarval settlement candidates. Especially, compound 1 is the strongest antifouling compound in nature until now which showed highly potent activity with picomolar level (EC 17.5 pM) and a very safety and high therapeutic ratio (LC/EC 1200). This represents an effective non-toxic, anti-larval settlement structural class of promising antifouling lead compound. [ABSTRACT FROM AUTHOR]

Identification of marine invertebrate larvae using morphological characters is laborious and complicated by phenotypic plasticity. Balanus amphitrite is a dominant barnacle, important in the context of intertidal ecology and biofouling of manmade structures. Morphological identification of barnacle larval forms in a mixed population is difficult because of their intricacy and similarity in size, shape and developmental stages. We report the development and application of a nucleic acid-based Polymerase Chain Reaction (PCR) method for the specific identification of the barnacle, B. amphitrite, from the heterogeneous zooplankton sample. This method is reliable and accurate thereby overcoming taxonomic ambiguity. Sequence alignment of the 18S rRNA gene region of selected species of barnacles allowed the design of B. amphitrite-specific PCR primers. Assay specificity was evaluated by screening DNA obtained from selected species of barnacles. The oligonucleotide primers used in the study flanked a 1600 bp region within the 18S rRNA gene. The primer is specific and can detect as few as 10 individuals of B. amphitrite larvae spiked in a background of ~186 mg of zooplankton. This technique facilitates accurate identification and the primer can be used as a marker for enumeration of B. amphitrite larvae in the plankton. [ABSTRACT FROM PUBLISHER]

Purpose - The purpose of this paper is to reduce the amount of copper in antifouling paints by using eugenol as an additive. Biofouling leads to deterioration of any submerged material. The most widespread method for control is the application of cuprous oxide antifouling paints which are toxic. First of all, the paper describes the effect of eugenol on larvae of Balanus amphitrite (fouling organism) under laboratory conditions and then the preparation, application and performance of different types of antifouling paints in field trials. Design/methodology/approach - Three types of soluble matrix antifouling paints were prepared with different pigments. The first one containing 16 per cent v/v copper, the second with 1.6 per cent copper and the third with 1.6 per cent copper + 2 per cent eugenol. Findings - After 12 months of immersion in Mar del Plata harbour paints containing 1.6 per cent copper + eugenol and 16 per cent copper were the most effective. Although these formulations showed a similar performance, copper + eugenol-based paint contains 90 per cent lesser copper than a traditional copper-based formulation. Originality/value - The use of antifouling paints with copper + eugenol combination as pigment is a promising alternative due to its performance, low cost and reduction in copper leaching to environment. [ABSTRACT FROM AUTHOR]

Ulva sp. and Gracilaria sp. were found colonizing shell surfaces of an acorn barnacle, Megabalanus tintinnabulum. However, this association was not noticed in the case of Balanus amphitrite, which was a co-inhabitant. Such a difference in intergeneric interactions with the algae was examined in laboratory experiments. For this, the influence that extracts of algae, extracts of algae-associated bacteria and natural leachants from M. tintinnabulum exerted on cyprid metamorphosis of B. amphitrite was examined. Extracts of algae and associated bacteria showed no effect on the metamorphosis of B. amphitrite. This may be attributed to absence of cue-specific sugars in the exopolysaccharides and culture supernatants of bacteria. On the other hand, natural leachants of M. tintinnabulum, which showed the presence of N-acetyl- d-glucosamine, a known algal spore attractant, also inhibited metamorphosis of B. amphitrite. Thus, hosting specific epibionts could have important roles in the segregation of barnacle population. [ABSTRACT FROM AUTHOR]

Testing of new coatings to control fouling frequently involves single-species laboratory bioassays. Barnacle cyprids are among the most widely used model organisms in marine biofouling research, and surfaces that inhibit their settlement are considered to be promising candidates for new coating concepts. An analysis of motility parameters (mean velocity and swimming area coefficient) and distribution of cyprids ofBalanus amphitritein different swimming regions in the vicinity of model surfaces (self-assembled monolayers) is presented. The data are correlated with the settlement preferences of cyprids on these surfaces. Cyprids were predominantly found in interfacial regions and the transition frequencies between swimming regions of different depths were determined. [ABSTRACT FROM AUTHOR]

Biofilm microbial communities play an important role in the larval settlement response of marine invertebrates. However, the underlying mechanism has yet to be resolved, mainly because of the uncertainties in characterizing members in the communities using traditional 16S rRNA gene-based molecular methods and in identifying the chemical signals involved. In this study, pyrosequencing was used to characterize the bacterial communities in intertidal and subtidal marine biofilms developed during two seasons. We revealed highly diverse biofilm bacterial communities that varied with season and tidal level. Over 3,000 operational taxonomic units with estimates of up to 8,000 species were recovered in a biofilm sample, which is by far the highest number recorded in subtropical marine biofilms. Nineteen phyla were found, of which Cyanobacteria and Proteobacteria were the most dominant one in the intertidal and subtidal biofilms, respectively. Apart from these, Actinobacteria, Bacteroidetes, and Planctomycetes were the major groups recovered in both intertidal and subtidal biofilms, although their relative abundance varied among samples. Full-length 16S rRNA gene clone libraries were constructed for the four biofilm samples and showed similar bacterial compositions at the phylum level to those revealed by pyrosequencing. Laboratory assays confirmed that cyrids of the barnacle Balanus amphitrite preferred to settle on the intertidal rather than subtidal biofilms. This preference was independent of the biofilm bacterial density or biomass but was probably related to the biofilm community structure, particularly, the Proteobacterial and Cyanobacterial groups. [ABSTRACT FROM AUTHOR]

Barnacles are major sessile components of the intertidal areas worldwide, and also one of the most dominant fouling organisms in fouling communities. Larval settlement has a crucial ecological effect not only on the distribution of the barnacle population but also intertidal community structures. However, the molecular mechanisms involved in the transition process from the larval to the juvenile stage remain largely unclear. In this study, we carried out comparative proteomic profiles of stage II nauplii, stage VI nauplii, cyprids, and juveniles of the barnacle Balanus amphitrite using label-free quantitative proteomics, followed by the measurement of the gene expression levels of candidate proteins. More than 700 proteins were identified at each stage; 80 were significantly up-regulated in cyprids and 95 in juveniles vs other stages. Specifically, proteins involved in energy and metabolism, the nervous system and signal transduction were significantly up-regulated in cyprids, whereas proteins involved in cytoskeletal remodeling, transcription and translation, cell proliferation and differentiation, and biomineralization were up-regulated in juveniles, consistent with changes associated with larval metamorphosis and tissue remodeling in juveniles. These findings provided molecular evidence for the morphological, physiological and biological changes that occur during the transition process from the larval to the juvenile stages in B. amphitrite. [ABSTRACT FROM AUTHOR]

Barnacles are major sessile components of the intertidal areas worldwide, and also one of the most dominant fouling organisms in fouling communities. Larval settlement has a crucial ecological effect not only on the distribution of the barnacle population but also intertidal community structures. However, the molecular mechanisms involved in the transition process from the larval to the juvenile stage remain largely unclear. In this study, we carried out comparative proteomic profiles of stage II nauplii, stage VI nauplii, cyprids, and juveniles of the barnacle Balanus amphitrite using label-free quantitative proteomics, followed by the measurement of the gene expression levels of candidate proteins. More than 700 proteins were identified at each stage; 80 were significantly up-regulated in cyprids and 95 in juveniles vs other stages. Specifically, proteins involved in energy and metabolism, the nervous system and signal transduction were significantly up-regulated in cyprids, whereas proteins involved in cytoskeletal remodeling, transcription and translation, cell proliferation and differentiation, and biomineralization were up-regulated in juveniles, consistent with changes associated with larval metamorphosis and tissue remodeling in juveniles. These findings provided molecular evidence for the morphological, physiological and biological changes that occur during the transition process from the larval to the juvenile stages in B. amphitrite. [ABSTRACT FROM AUTHOR]

This work presents novel mapping of the structure and crystallography of the shell of the barnacle Balanus amphitrite. Calcium carbonate in the form of hexagonal calcite was observed, with fine crystallites on the order of 1 μm in diameter forming clusters of similar orientation. While no apparent preferred orientation was measured in the parietal shell cross section, the base plate shell cross section revealed a preference for the alignment of the 〈0001〉 direction at the exterior of the shell, rotating by 90° toward the center of the base plate cross-section. These maps will be used to construct image-based models of the barnacle shell for further study of mechanical response. [ABSTRACT FROM AUTHOR]

Biofouling results in tremendous economic losses to maritime industries around the world. A recent global ban on the use of organotin compounds as antifouling agents has further raised demand for safe and effective antifouling compounds. In this study, 49 secondary metabolites, including diterpenoids, steroids, and polyketides, were isolated from soft corals, gorgonians, brown algae, and fungi collected along the coast of China, and their antifouling activity was tested against cyprids of the barnacle Balanus ( Amphibalanus) amphitrite. Twenty of the compounds were found to inhibit larval settlement significantly at a concentration of 25 μg ml. Two briarane diterpenoids, juncin O ( 2) and juncenolide H ( 3), were the most promising non-toxic antilarval settlement candidates, with EC values less than 0.13 μg ml and a safety ratio (LC/EC) higher than 400. A preliminary structure-activity relationships study indicated that both furanon and furan moieties are important for antifouling activity. Intriguingly, the presence of hydroxyls enhanced their antisettlement activity. [ABSTRACT FROM AUTHOR]

Studies on the settlement and the subsequent recruitment of intertidal organisms are crucial steps in understanding their population structure in a particular bioregion. However, studying the recruitment of intertidal organisms such as barnacles, with two-phase life cycle, and understanding the determinants of recruitment is a complex problem. Many processes which operate during the pelagic pre-settlement phase and benthic post-settlement phase at different spatial and temporal scales can determine the fate of barnacle populations. In this study, observations were carried out on settlement and recruitment of the intertidal barnacle Balanus amphitrite from a tropical environment influenced by monsoons at spatial and temporal scales. Settlement and recruitment both showed significant temporal variations. In general, settlement and recruitment was lower during the monsoon season except during monsoon breaks. Consistency in settlement and recruitment was mostly observed during the pre-monsoon season, and it coincided with peaks in larval abundance observed during the same season. In general, settlement remained more or less similar on different types of substrata, whereas recruitment differed significantly. Temporal variations observed in settlement and recruitment of barnacles could be due to the influence of monsoons in this region, which leads to seasonal fluctuations in physical factors such as local hydrodynamics, that influence the retention and dispersal of larvae in the adult habitats, and could also be due to variations in the availability of food for the larvae and the settled populations. [ABSTRACT FROM PUBLISHER]

A novel configuration, consisting of two apposing surfaces bounding a vertical water column, is presented and evaluated for settlement assays using cyprids ofBalanus amphitrite. Assays were conducted on planar surfaces, ranging from hydrophobic polystyrene to hydrophilic glass and including CH3- and NH3+-terminated self-assembled monolayers (SAMs). Identical apposing surfaces generated settlement rates comparable to those obtained in prior studies, while a choice assay yielded consistent results, with individual replicates each indicating the preferred surface for settlement. As gravity favours contact with the lower apposing surface, cyprids trapped at the air/water interface settled on or around the perimeter where the water column meets the lower substratum. These cyprids are capable of selecting a settlement location and are thus not lost to the assay. The assay geometry lends itself to assessing cyprid exploration and settlement on planar surfaces with chemical patterning, including relief microstructures, without using a confining material or requiring the coating of a three-dimensional well. [ABSTRACT FROM PUBLISHER]

The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling. [ABSTRACT FROM AUTHOR]

Abstract: Five new cembrane-based diterpenoids, namely pavidolides A–E (1–5) were isolated from the marine soft coral Sinularia pavida, together with sarcophytin and chatancin. The structures of new compounds were determined on the basis of extensive spectroscopic data analysis. Pavidolide B (2) possesses an unprecedented 6,5,7-tricarbocyclic nucleus, whereas pavidolide C (3) is characteristic of an unusual C-5 and C-9 conjuncted cembranoid. Pavidolides C and D showed moderate antifouling activity against the larval settlement of barnacle Balanus amphitrite, while pavidolides B and C exhibited inhibitory activity against the human leukemia cell line HL-60. [Copyright &y& Elsevier]

The barnacle Balanus ( = Amphibalanus) amphitrite is a major marine fouling animal. Understanding the molecular mechanism of larval settlement in this species is critical for anti-fouling research. In this study, we cloned one isoform of p38 MAPK (Bar-p38 MAPK) from this species, which shares the significant characteristic of containing a TGY motif with other species such as yeast, Drosophila and humans. The activation of p38 MAPK was detected by an antibody that recognizes the conserved dual phosphorylation sites of TGY. The results showed that phospho-p38 MAPK (pp38 MAPK) was more highly expressed at the cyprid stage, particularly in aged cyprids, in comparison to other stages, including the nauplius and juvenile stages. Immunostaining showed that Bar-p38 MAPK and pp38 MAPK were mainly located at the cyprid antennules, and especially the third and fourth segments, which are responsible for substratum exploration during settlement. The expression and localization patterns of Bar-p38 MAPK suggest its involvement in larval settlement. This postulation was also supported by the larval settlement bioassay with the p38 MAPK inhibitor SB203580. Behavioral analysis by live imaging revealed that the larvae were still capable of exploring the surface of the substratum after SB203580 treatment. This shows that the effect of p38 MAPK on larval settlement might be by regulating the secretion of permanent proteinaceous substances. Furthermore, the level of pp38 MAPK dramatically decreased after full settlement, suggesting that Bar-p38 MAPK maybe plays a role in larval settlement rather than metamorphosis. Finally, we found that Bar-p38 MAPK was highly activated when larvae confronted extracts of adult barnacle containing settlement cues, whereas larvae pre-treated with SB203580 failed to respond to the crude adult extracts. [ABSTRACT FROM AUTHOR]

The barnacle Balanus amphitrite is a globally distributed marine crustacean and has been used as a model species for intertidal ecology and biofouling studies. Its life cycle consists of seven planktonic larval stages followed by a sessile juvenile/adult stage. The transitional processes between larval stages and juveniles are crucial for barnacle development and recruitment. Although some studies have been conducted on the neuroanatomy and neuroactive substances of the barnacle, a comprehensive understanding of neuropeptides and peptide hormones remains lacking. To better characterize barnacle neuropeptidome and its potential roles in larval settlement, an in silico identification of putative transcripts encoding neuropeptides/peptide hormones was performed, based on transcriptome of the barnacle B. amphitrite that has been recently sequenced. Potential cleavage sites and structure of mature peptides were predicted through homology search of known arthropod peptides. In total, 16 neuropeptide families/subfamilies were predicted from the barnacle transcriptome, and 14 of them were confirmed as genuine neuropeptides by Rapid Amplification of cDNA Ends. Analysis of peptide precursor structures and mature sequences showed that some neuropeptides of B. amphitrite are novel isoforms and shared similar characteristics with their homologs from insects. The expression profiling of predicted neuropeptide genes revealed that pigment dispersing hormone, SIFamide, calcitonin, and B-type allatostatin had the highest expression level in cypris stage, while tachykinin-related peptide was down regulated in both cyprids and juveniles. Furthermore, an inhibitor of proprotein convertase related to peptide maturation effectively delayed larval metamorphosis. Combination of real-time PCR results and bioassay indicated that certain neuropeptides may play an important role in cypris settlement. Overall, new insight into neuropeptides/peptide hormones characterized in this study shall provide a platform for unraveling peptidergic control of barnacle larval behavior and settlement process. [ABSTRACT FROM AUTHOR]

Barnacles adhere permanently to surfaces by secretingand curinga thin interfacial adhesive underwater. Here, we show that the acornbarnacle Balanus amphitriteadheresby a two-step fluid secretion process, both contributing to adhesion.We found that, as barnacles grow, the first barnacle cement secretion(BCS1) is released at the periphery of the expanding base plate. Subsequently,a second, autofluorescent fluid (BCS2) is released. We show that secretionof BCS2 into the interface results, on average, in a 2-fold increasein adhesive strength over adhesion by BCS1 alone. The two secretionsare distinguishable both spatially and temporally, and differ in morphology,protein conformation, and chemical functionality. The short time windowfor BCS2 secretion relative to the overall area increase demonstratesthat it has a disproportionate, surprisingly powerful, impact on adhesion.The dramatic change in adhesion occurs without measurable changesin interface thickness and total protein content. A fracture mechanicsanalysis suggests the interfacial material’s modulus or workof adhesion, or both, were substantially increased after BCS2 secretion.Addition of BCS2 into the interface generates highly networked amyloid-likefibrils and enhanced phenolic content. Both intertwined fibers andphenolic chemistries may contribute to mechanical stability of theinterface through physically or chemically anchoring interface proteinsto the substrate and intermolecular interactions. Our experimentspoint to the need to reexamine the role of phenolic components inbarnacle adhesion, long discounted despite their prevalence in structuralmembranes of arthropods and crustaceans, as they may contribute tochemical processes that strengthen adhesion through intermolecularcross-linking. [ABSTRACT FROM AUTHOR]

Abstract: Amphibalanus (=Balanus) amphitrite is a tropical/sub-tropical barnacle species which is naturally exposed to sea temperatures of 16–27 °C throughout its larval development. An established technique widely employed by investigators of A. amphitrite settlement involves the storage of cyprids at 4–6 °C prior to their use in bioassays. Our study focuses on the effects on ageing temperature on settlement and discrimination of A. amphitrite larvae. Using cyprids aged at 2 temperatures, 6° and 23 °C, we confirmed the general trend in the literature that young d0 cyprids of this species do not appear competent to settle. Performing cyprid settlement assays at 20, 25 or 28 °C, we observed that the proportion of settled cyprids when incubated at 28 °C was greater than that of cyprid incubated at 25 °C and 20 °C. Settlement rates of cyprids aged at 6 °C and 23 °C increased relative to age from d1 to d10 irrespective of temperature. Cyprids lost the ability to undergo attachment and metamorphosis at d14 or d15 when aged at 6 °C or 23 °C respectively. In the choice assays executed in this study, cyprids generally chose to settle on adult extract-treated areas. But localised settlement on adult extract-treated areas decreased with time. Choice settlement assays at set age intervals during the cypris larval stage showed that cyprids were able to discriminate between con- and allospecific adult extracts. The duration and the temperature of cyprids storage influenced selectivity. [Copyright &y& Elsevier]

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants over the last three decades, and are now ubiquitous in the marine environment. While the harmful effects of PBDEs on the abnormal development and reproductive impairment in mammals and fish are well documented, the effects on marine invertebrates remain virtually unknown. Using three model intertidal species accross three phyla, including the polychaete Hydroides elegans (Phylum Annelida), the gastropod Crepidula onyx (Phylum Mollusca), and the barnacle Balanus amphitrite (Phylum Arthopoda), this study demonstrated that (a) chronic exposure to BDE-47 (at spiking concentrations up to 1000 ng L-1) throughout the entire larval stage did not affect settlement, development or growth of all three species per se, despite bioaccumulation was clearly evident (measured body burden ranging from approximately 7000 to 13 000 ng BDE-47 g-1 lipid), and (b) BDE-47, at measured concentrations of 15 and 113 ng g-1 lipid, reduced the bacterial abundance in biofilms and resulted in a concomitant change in larval settlement pattern of all the model intertidal species across three phyla. [ABSTRACT FROM AUTHOR]

Amphiphilic copolymers containing different amounts of poly(ethylene glycol)-fluoroalkyl acrylate and polysiloxane methacrylate units were blended with a poly(dimethyl siloxane) (PDMS) matrix in different proportions to investigate the effect of both copolymer composition and loading on the biological performance of the coatings. Laboratory bioassays revealed optimal compositions for the release of sporelings of Ulva linza, and the settlement of cypris larvae of Balanus amphitrite. The best-performing coatings were subjected to field immersion tests. Experimental coatings containing copolymer showed significantly reduced levels of hard fouling compared to the control coatings (PDMS without copolymer), their performance being equivalent to a coating based on Intersleek 700™. XPS analysis showed that only small amounts of fluorine at the coating surface were sufficient for good antifouling/fouling-release properties. AFM analyses of coatings under immersion showed that the presence of a regular surface structure with nanosized domains correlated with biological performance. [ABSTRACT FROM AUTHOR]

Stalked and acorn barnacles (Cirripedia Thoracica) have a complex life cycle that includes a free-swimming nauplius larva, a cypris larva and a permanently attached sessile juvenile and adult barnacle. The barnacle cyprid is among the most highly specialized of marine invertebrate larvae and its settlement biology has been intensively studied. By contrast, surprisingly few papers have dealt with the critical series of metamorphic events from cementation of the cyprid to the substratum until the appearance of a suspension feeding juvenile. This metamorphosis is both ontogenetically complex and critical to the survival of the barnacle. Here we use video microscopy to present a timeline and description of morphological events from settled cyprid to juvenile barnacle in the model species Balanus amphitrite, representing an important step towards both a broader understanding of the settlement ecology of this species and a platform for studying the factors that control its metamorphosis. Metamorphosis in B. amphitrite involves a complex sequence of events: cementation, epidermis separation from the cypris cuticle, degeneration of cypris musculature, rotation of the thorax inside the mantle cavity, building of the juvenile musculature, contraction of antennular muscles, raising of the body, shedding of the cypris cuticle, shell plate and basis formation and, possibly, a further moult to become a suspension feeding barnacle. We compare these events with developmental information from other barnacle species and discuss them in the framework of barnacle settlement ecology. [ABSTRACT FROM AUTHOR]

A series of 10 intra-laboratory bioassays and an inter-laboratory assay involving nine laboratories, were performed to validate the use of Amphibalanus amphitrite larvae as test organisms for ecotoxicological studies. The standardization protocol utilized Cadmium Nitrate as a reference toxicant and larval immobilization (after 24 and 48h) as the end-point. The statistical data analysis showed high homogeneity in intra-laboratory EC50 values among bioassay repetitions (coefficient of variation (CV)=15.8% after 24h and 16.9% after 48h); also CV values obtained in inter laboratory comparison exercise were not higher than the 50%, variation coefficient mentioned in the ISO standards for the precision of inter-laboratory assays. The results demonstrated that the protocol implemented for the balanus larval bioassay comply with the international standards both for intra- and inter-laboratory precision. [Copyright &y& Elsevier]

Abstract: Exploration of substrata prior to cementation and metamorphosis is a critical step in the life cycle of Cirripedes. To this end, barnacle cyprid larvae have evolved sophisticated sensory mechanisms to investigate chemical as well as topographical composition of the substratum. Since barnacles are major biofoulers, the sensory structures of Balanus amphitrite cyprids have been described in detail using ultrastructural techniques like SEM and TEM. Settlement assays have shown that cyprid settlement is deterred by adrenergic receptor agonists and antagonists, yet the exact location of these receptors on cyprid sensory structures is unknown. Here, using a fluorescent adrenergic receptor antagonist (Bodipy FL-Prazosin) with the aid of confocal microscopy we describe the putative locations of adrenergic-like receptors in B. amphitrite cyprids. Our results indicate that postaxial setae 2 and 3 of the 2nd and 3rd segments respectively, the sub-terminal setae of the 4th antennular segment and the cement gland secretory cells seem to bear the adrenergic-like receptors. Using optical and electron microscopy we further studied the effects of noradrenaline (NA), an adrenergic receptor agonist, on cyprid settlement and metamorphosis. NA treated cyprids forgo the typical settlement behavior and metamorphose into juvenile barnacles without cementing to the substratum. Cyprid-barnacle metamorphosis in NA treated cyprids is also prolonged as compared to untreated barnacles. The key role played by adrenergic-like receptors during surface sensing, cementation and metamorphosis is discussed. [Copyright &y& Elsevier]

Many barnacle species are gregarious and their cypris larvae display a remarkable ability to explore surfaces before committing to permanent attachment. The chemical cue to gregarious settlement behaviour - the settlement-inducing protein complex (SIPC) - Is an α2-macroglobulin-like glycoprotein. This cuticular protein may also be involved in cyprid reversible adhesion if its presence is confirmed in footprints of adhesive deposited during exploratory behaviour, which increase the attractiveness of surfaces and signal other cyprids to settle. The full-length open-reading frame of the SIPC gene encodes a protein of 1547 amino acids with seven potential N-glycosylation sites. In this study on Balanus amphitrite, glycan profiling of the SIPC via hydrophlllc interaction liquid chromatography with fluorescence detection (HILIC-fluorescence) provided evidence of predominantly high mannose glycans (M2-9), with the occurrence of monofucosylated oligomannose glycans (F(6)M2-4) in lower proportions. The high mannose glycosylation found supports previous observations of an interaction with mannose-blnding lectins and exogenous mannose increasing settlement in B. amphitrite cypris larvae. Transmission electron microscopy of the deglycosylated SIPC revealed a multl-lobed globular protein with a diameter of ~8nm. Obtaining a complete structural characterisation of the SIPC remains a goal that has the potential to inspire solutions to the age-old problem of barnacle fouling. [ABSTRACT FROM AUTHOR]