Effects of poly-ether B on proteome and phosphoproteome expression in biofouling Balanus amphitrite cyprids

Biofouling is ubiquitous in marine environments, and the barnacle Balanus amphitrite is one of the most recalcitrant and aggressive biofoulers in tropical waters. Several natural antifoulants that were claimed to be non-toxic have been isolated in recent years, although the mechanism by which they inhibit fouling is yet to be investigated. Poly-ether B has shown promise in the non-toxic inhibition of larval barnacle attachment. Hence, in this study, multiplex two-dimensional electrophoresis (2-DE) was applied in conjunction with mass spectrometry to investigate the effects of poly-ether B on barnacle larvae at the molecular level. The cyprid proteome response to poly-ether B treatment was analyzed at the total proteome and phosphoproteome levels, with 65 protein and 19 phosphoprotein spots found to be up- or down-regulated. The proteins were found to be related to energy-metabolism, oxidative stress, and molecular chaperones, thus indicating that poly-ether B may interfere with the redox-regulatory mechanisms governing the settlement of barnacle larvae. The results of this study demonstrate the usefulness of the proteomic technique in revealing the working mechanisms of antifouling compounds.     

Response of larval barnacle proteome to CO(2)-driven seawater acidification

The majority of benthic marine invertebrates have a complex life cycle, during which the pelagic larvae select a suitable substrate, attach to it, and then metamorphose into benthic adults. Anthropogenic ocean acidification (OA) is postulated to affect larval metamorphic success through an altered protein expression pattern (proteome structure) and post-translational modifications. To test this hypothesis, larvae of an economically and ecologically important barnacle species Balanus amphitrite, were cultured from nauplius to the cyprid stage in the present (control) and in the projected elevated concentrations of CO₂ for the year 2100 (the OA treatment). Cyprid response to OA was analyzed at the total proteome level as well as two protein post-translational modification (phosphorylation and glycosylation) levels using a 2-DE based proteomic approach. The cyprid proteome showed OA-driven changes. Proteins that were differentially up or down regulated by OA come from three major groups, namely those related to energy-metabolism, respiration, and molecular chaperones, illustrating a potential strategy that the barnacle larvae may employ to tolerate OA stress. The differentially expressed proteins were tentatively identified as OA-responsive, effectively creating unique protein expression signatures for OA scenario of 2100. This study showed the promise of using a sentinel and non-model species to examine the impact of OA at the proteome level.     

Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite

The barnacle, Balanus amphitrite, is one of the primary model organisms for rocky-shore ecology studies and biofouling research. This barnacle species has a complex life cycle during which the swimming nauplius molts six times and transforms into a cyprid stage. Cyprids must attach to a surface to metamorphose into a juvenile barnacle. To clarify the overall profile of protein expression during larval development and metamorphosis, 2-DE was used to compare the proteome of the nauplius, the swimming cyprid, the attached cyprid, and the metamorphosed cyprid. The proteome of the swimming cyprid was distinctly different from that of other life stages and had about 400 spots. The proteomes of the attached and metamorphosed cyprids were similar with respect to major proteins but had significantly lower numbers of spots compared to that of swimming larval stages. Obviously, synthesis of most proteins from swimming cyprids was switched off after attachment and metamorphosis. Our advanced MS analysis (MALDI-TOF/TOF MS/MS) allowed us to identify the proteins that were differentially and abundantly expressed in the swimming cyprid. These proteins included signal transduction proteins (adenylate cyclase and calmodulin) and juvenile hormone binding proteins. In summary, for the first time, we have analyzed the global protein expression pattern of fouling marine invertebrate larvae during metamorphosis. Our study provides new insights into the mechanisms of barnacle larval metamorphosis and also provides a foundation for exploring novel targets for antifouling treatments.     

Settlement behaviour of marine invertebrate larvae measured by EthoVision 3.0

Submerged marine surfaces are rapidly colonized by fouling organisms. Current research is aimed at finding new, non-toxic, or at least environmentally benign, solutions to this problem. Barnacles are a major target organism for such control as they constitute a key component of the hard fouling community. A range of standard settlement assays is available for screening test compounds against barnacle cypris larvae, but they generally provide little information on mechanism(s) of action. Towards this end, a quick and reliable video-tracking protocol has been developed to study the behaviour of the cypris larvae of the barnacle, Balanus amphitrite, at settlement. EthoVision 3.0 was used to track individual cyprids in 30-mm Petri dishes. Experiments were run to determine the optimal conditions vis-a-vis acclimation time, tracking duration, number of replicates, temperature and lighting. A protocol was arrived at involving a two Petri dish system with backlighting, and tracking over a 5-min period after first acclimating the cyprids to test conditions for 2 min. A minimum of twenty replicates was required to account for individual variability in cyprid behaviour from the same batch of larvae. This methodology should be widely applicable to both fundamental and applied studies of larval settlement and with further refinements, to that of smaller fouling organisms such as microalgae and bacteria.     

Isethionic acid and floridoside isolated from the red alga, Grateloupia turuturu, inhibit settlement of Balanus amphitrite cyprid larvae

Isethionic acid (2-hydroxyethane sulfonic acid) and floridoside (2-O-alpha-D-galactopyranosylglycerol) were extracted from the red alga, Grateloupia turuturu, and tested for anti-settlement activity against cyprid larvae of the tropical barnacle, Balanus amphitrite and for their toxicity to nauplius larvae. Isethionic acid was active for anti-settlement but had the disadvantage of being toxic to nauplius larvae. Floridoside was a potent inhibitor of cyprid settlement at non-toxic concentrations to nauplii (0.01 mg ml(-1)).     

Settlement Behaviour of Marine Invertebrate Larvae Measured by EthoVision 3.0

Submerged marine surfaces are rapidly colonized by fouling organisms. Current research is aimed at finding new, non-toxic, or at least environmentally benign, solutions to this problem. Barnacles are a major target organism for such control as they constitute a key component of the hard fouling community. A range of standard settlement assays is available for screening test compounds against barnacle cypris larvae, but they generally provide little information on mechanism(s) of action. Towards this end, a quick and reliable video-tracking protocol has been developed to study the behaviour of the cypris larvae of the barnacle, Balanus amphitrite, at settlement. EthoVision 3.0 was used to track individual cyprids in 30-mm Petri dishes. Experiments were run to determine the optimal conditions vis-à-vis acclimation time, tracking duration, number of replicates, temperature and lighting. A protocol was arrived at involving a two Petri dish system with backlighting, and tracking over a 5-min period after first acclimating the cyprids to test conditions for 2 min. A minimum of twenty replicates was required to account for individual variability in cyprid behaviour from the same batch of larvae. This methodology should be widely applicable to both fundamental and applied studies of larval settlement and with further refinements, to that of smaller fouling organisms such as microalgae and bacteria.     

Inhibition of Settlement by Larvae of Balanus amphitrite and Ciona intestinalis by a Surface-Colonizing Marine Bacterium

In an attempt to isolate bacteria with inhibitory effects against settlement by larvae of sessile invertebrates, 40 marine bacterial isolates were screened for effects against laboratory-reared barnacle larvae (Balanus amphitrite) and ascidian larvae (Ciona intestinalis). Five isolates displayed non-pH-dependent inhibitory effects against the larvae. The initial characterization of a toxic component released from an isolate, designated D2 (CCUG 26757), and its effect on laboratory-reared barnacle and ascidian larvae were studied. D2 is a facultative, anaerobic, gram-negative bacterium isolated from the surface of C. intestinalis from waters off the Swedish west coast at a depth of 10 m. Results suggest that the toxic component is released by D2 during the stationary phase. Aged biofilms were more toxic to the larvae than unaged films. The biologically active compound was in the supernatant of D2 and was heat stable and <500 Da in molecular mass. No evidence of protein or peptide moieties was found. On the basis of two phase and chromatography separations, the component is polar and neutral and contains or binds to carbohydrate moieties. Metaperiodate treatment increased toxicity; undiluted supernatant from a 24-h growth culture of D2 killed barnacle and ascidian larvae within a few hours of exposure, whereas after metaperiodate treatment, the larvae were killed in approximately 30 min.     

Isethionic Acid and Floridoside Isolated from the Red Alga,Grateloupia turuturu,Inhibit Settlement of Balanus amphitrite Cyprid Larvae

Isethionic acid (2-hydroxyethane sulfonic acid) and floridoside (2-O-α-D-galactopyranosylglycerol) were extracted from the red alga, Grateloupia turuturu, and tested for anti-settlement activity against cyprid larvae of the tropical barnacle, Balanus amphitrite and for their toxicity to nauplius larvae. Isethionic acid was active for anti-settlement but had the disadvantage of being toxic to nauplius larvae. Floridoside was a potent inhibitor of cyprid settlement at non-toxic concentrations to nauplii (0.01 mg ml^?1).     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.     

Are the two physiological races of Pollicipes polymerus (Cirripedia) genetically divided along the California coast?

Abstract. Genetic work on numerous species with planktonically dispersing larvae has demonstrated gene flow throughout populations along the California coast, despite steep environmental gradients around Point Conception. Previous ecological work on a planktonically dispersing intertidal barnacle, Pollicipes polymerus , demonstrated that this species is divided into two physiological races near Point Conception, at Point Latigo. The northern race broods when water temperature either warms or cools to 14°C and the southern race broods as water warms to 20°C. I examined 8 polymorphic allozyme loci from five populations between Santa Cruz and San Diego to test if the two physiological races are genetically divided. No genetic differences were found over this geographic range, supporting a previous molecular study. These results suggest that reproductive differences between the two races of this stalked barnacle may result from plasticity in brooding activity.     

Bacteria immobilised in Gels: Improved methodologies for antifouling and biocontrol applications

A range of bacteria, including the marine bacterium Pseudoalteromonas tunicata which produces antifouling compounds, and Escherichia coli were used to investigate methods for immobilising bacteria in gels. Different types of matrices were screened using the survival of barnacle nauplii as a bioassay. A Dupont? polyvinylalcohol (PVOH) 10% gel was found to be the optimal matrix. This non-toxic gel remained stable in seawater while allowing for an outflux of active biological compounds from the bacterial cells. The presence of active bacterial cells in the matrix was tested by CTC-staining, green fluorescent protein (GFP) expressing bacteria and a barnacle larvae bioassay. The Dupont? PVOH 10% gels containing P. tunicata cells were inhibitory against larvae for a period of up to 2 weeks. In further studies using gels containing immobilised bacteria, the E. coli strain C600 was employed based on its cell size, stress resistance and the fact that a plasmid for the expression of GFP could be transferred and maintained in the cells. Immobilised E. Coli cells maintained their viability in the Dupont? PVOH 10% gels for as long as 2 months, and the life-span of these "biologically active"; gels was increased to more than 2 months by the incorporation of small beads into the gels. The results indicate that bacteria can be immobilised in coatings for periods of time consistent with the needs of some antifouling and antibacterial applications.     

Nontoxic piperamides and their synthetic analogues as novel antifouling reagents

Bioassay-guided isolation of an acetone extract from a terrestrial plant Piper betle produced four known piperamides with potent antifouling (AF) activities, as evidenced by inhibition of settlement of barnacle cypris larvae. The AF activities of the four piperamides and 15 synthesized analogues were compared and their structure–activity relationships were probed. Among the compounds, piperoleine B and 1-[1-oxo-7-(3′,4′-methylenedioxyphenyl)-6E-heptenyl]-piperidine (MPHP) showed strong activity against settlement of cyprids of the barnacle Balanus amphitrite, having EC₅₀ values of 1.1?±?0.3 and 0.5?±?0.2?μg?ml^?1, respectively. No toxicity against zebra fish was observed following incubation with these two compounds. Besides being non-toxic, 91% of piperoleine B-treated cyprids and 84% of MPHP-treated cyprids at a concentration of 100?μM completed normal metamorphosis in recovery bioassays, indicating that the anti-settlement effect of these two compounds was reversible. Hydrolysis and photolysis experiments indicated that MPHP could be decomposed in the marine environment. It is concluded that piperamides are promising compounds for use in marine AF coatings.     

Variation in barnacle recruitment over small scales: larval predation by adults and maintenance of community pattern

Over small spatial scales, variation in the density of settlers of benthic sessile species is the result of interactions among larval behavior, local hydrodynamic conditions, and the physical, chemical and biological characteristics of the benthic habitat. It has been shown repeatedly that adult benthic filter-feeders can consume larvae of their own and other species, but their effects on the distribution and abundance of recruits have rarely been demonstrated under natural conditions in the field, particularly on hard substrata. Here we experimentally quantified the effect of the large intertidal barnacle, Semibalanus cariosus (Pallas), on the density of recruits of three common barnacle species. The experiments were conducted at the peak of the barnacle recruitment season over three successive years, on the west coast of San Juan Island, Washington. A persistent and well documented community pattern in the mid intertidal zone of the study site is a sparse bed of adult S. cariosus with bare rock spaces essentially devoid of small barnacles among the large individuals. Field experiments consisted of small areas from which either all adult S. cariosus were killed leaving the shells attached to the rock, or live adult barnacles were left intact. Our results showed that over small spatial scales of a few to tens of centimeters, the large barnacle S. cariosus can interfere and significantly reduce net settlement and recruitment of conspecific as well as other barnacle species. Between 65 and 100% reduction in settlement could be attributed to larval predation by adults, as implied by barnacle settlement patterns on different treatments and by the presence of nauplius larvae in cirri and stomach contents of S. cariosus. The negative effect on barnacle settlement was consistent between years of relatively low barnacle recruitment, which appears to be the most common situation at the study site, but it disappeared on a year of unusually high recruitment, when settling larvae seem to have swamped the filtration ability of adult S. cariosus. The different barnacle species displayed contrasting settlement patterns on bare rock and on the lateral shells of the large barnacles, which appear to be a result of differences in larval behavior. Comparisons against the relative availability of these substrata in the experimental plots suggested that larvae of different species sample the benthic microhabitat in very different ways.     

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.     

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.     

Development of a tissue cell culture bioassay for identifying mechanisms of inhibition of settlement of barnacle and tunicate larvae by surface‐colonizing marine bacteria

A marine bacterium D2 (CCUG 26757) isolated from a tunicate Ciona intestinalis specimen produced a low molecular weight component which inhibits barnacle and tunicate larvae and prevents their settlement on solid surfaces (Holmström et al., 1992). In order to perform chemical and structural analyses of the component independent of season, a bioassay, complementary to tests with invertebrate larvae was developed. This bioassay is based on tissue cell culture techniques and growth of the AGS cell line. It was previously shown that the toxic component is a stationary phase released product, which is heat stable and less than 500 Dalton in size (Holmström et al., 1992). Furthermore, it is not a peptide or a protein and metaperiodate treatment increases its toxicity to larvae indicating that it binds to or contains carbohydrate moieties. In this study, these results were confirmed by using the anchorage dependent human gastric adenocarcinoma (AGS) cell line as a bioassay. Fractionation of the D2 supernatant on a Sephadex G‐200 column and addition of different size fractions to the AGS tissue culture cells, showed that both a low and a high molecular weight fraction inhibited cell growth. Exposure of Balanus amphitrite and C. intestinalis larvae to the same fractions, showed that the low molecular weight fraction that inhibited growth of the cells corresponds to the component that inhibited larvae of both organisms. The high molecular weight fraction was found to inhibit larvae of B. amphitrite.     

Surface wettability as a determinant in the settlement of the barnacle Balanus Improvisus (DARWIN)

Several studies have shown that the initial surface wettability, is of importance in the settlement of macrofouling larvae such as barnacles, bryozoans and hydroids in the field as well as in laboratory assays. In this study we present results from laboratory assays using hydrophilic and hydrophobic polystyrene (PS) and cyprid larvae of Balanus improvisus (Darwin). The results obtained differ markedly from those reported for the barnacle Balanus amphitrite (Darwin), where a high surface wettability seemed to be preferred for settlement. Our results show that a surface with intermediary wettability (hydrophilic PS) reduced settlement by 38% as compared to surfaces of low wettability (hydrophobic PS) during an 8-day period. During the experiment, the wettability in the hydrophilic PS dishes was not significantly changed as measured by advancing contact angle with mQ water. Over an 8-day period wettability of the hydrophobic PS dishes approached that of the hydrophilic PS surfaces. We further conducted experiments with highly hydrophilic and highly hydrophobic methylsilane-treated glass surfaces with known chemistry. In this experiment, the settlement of cyprid larvae was completely inhibited by the high wettability surfaces. Contact angle measurements revealed that the wettability during the length of the experiment of the hydrophilic glass surfaces was not significantly altered. We conclude by these experiments that even an intermediate wettability can significantly affect the overall settlement success of the barnacle B. improvisus. The mechanism by which the settlement is impeded might be biologically mediated through the recognition by cyprid larvae of the molecular composition of the surface when the cyprid reverts to the settlement phase, i.e. when swimming behaviour is abandoned in favour of surface exploration, or it is mediated by physicochemical forces acting between the surface and the larval body or the larval antennules.     

Antifouling activity of simple synthetic isocyanides against larvae of the barnacle Balanus amphitrite

Twelve simple linear isocyanides were synthesized and examined for antifouling activity and toxicity against cyprid larvae of the barnacle, Balanus amphitrite. Larval settlement was inhibited, with EC50 values of 0.046-1.90 microg ml(-1), and they were much less toxic (LD50 values ranging over 21.28 microg ml(-1)) than CuSO4 (EC50 0.30 microg ml(-1) and LD50 2.95 microg ml(-1)). The data indicate that simple linear isocyanides are promising non-toxic antifouling agents.     

Evidence for the Involvement of p38 MAPK Activation in Barnacle Larval Settlement

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.