Effects of surface charge and Gibbs surface energy on the settlement behaviour of barnacle cyprids (Balanus amphitrite)

Gibbs surface energy has long been considered to be an important parameter in the design of fouling-resistant surfaces for marine applications. Rigorous testing of the hypothesis that settlement is related to Gibbs surface energy however has never been accomplished, due mainly to practical limitations imposed by the necessary combination of surface engineering and biological evaluation methods. In this article, the effects of surface charge and Gibbs surface energy on the settlement of cyprids of an important fouling barnacle, Balanus amphitrite, were evaluated. Settlement assays were conducted on a range of self-assembled monolayers (SAMs) (CH₃-, OH-, COOH-, N(CH₃)₃ ⁺-, NH₂-terminated), presented in gold-coated polystyrene well plates, varying in terms of their surface charge and Gibbs surface energy. Contrary to contemporary theory, settlement was not increased by high-energy surfaces, rather the opposite was found to be the case with cyprids settling in greater numbers on a low-energy CH₃- SAM compared to a high-energy OH- SAM. Settlement was also greater on negatively-charged SAMs, compared to neutral and positively-charged SAMs. These findings are discussed in the context of data drawn from surfaces that varied in multiple characteristics simultaneously, as have been used previously for such experiments. The finding that surface charge, rather than total surface energy, may be responsible for surface selection by cyprids, will have significant implications for the design of future fouling-resistant materials.     

Correlation between surface chemistry and settlement behaviour in barnacle cyprids (Balanus improvisus)

In laboratory-based biofouling assays, the influence of physico-chemical surface characteristics on barnacle settlement has been tested most frequently using the model organism Balanus amphitrite (= Amphibalanus amphitrite). Very few studies have addressed the settlement preferences of other barnacle species, such as Balanus improvisus (= Amphibalanus improvisus). This study aimed to unravel the effects of surface physico-chemical cues, in particular surface-free energy (SFE) and surface charge, on the settlement of cyprids of B. improvisus. The use of well-defined surfaces under controlled conditions further facilitates comparison of the results with recent similar data for B. amphitrite. Zero-day-old cyprids of B. improvisus were exposed to a series of model surfaces, namely self-assembled monolayers (SAMs) of alkanethiols with varying end-groups, homogenously applied to gold-coated polystyrene (PS) Petri dishes. As with B. amphitrite, settlement of cyprids of B. improvisus was influenced by both SFE and charge, with higher settlement on low-energy (hydrophobic) surfaces and negatively charged SAMs. Positively charged SAMs resulted in low settlement, with intermediate settlement on neutral SAMs of similar SFE. In conclusion, it is demonstrated that despite previous suggestions to the contrary, these two species of barnacle show similar preferences in response to SFE; they also respond similarly to charge. These findings have positive implications for the development of novel antifouling (AF) coatings and support the importance of consistency in substratum choice for assays designed to compare surface preferences of fouling organisms.     

Presence and distribution of serotonin immunoreactivity in the cyprids of the barnacle Balanus amphitrite

In this work, the presence and distribution of serotonin in the cyprid of the barnacle Balanus amphitrite were investigated by immunohistochemical methods. Serotonin-like immuno-reactive neuronal cell bodies were detected in the central nervous system only. Various clusters of immunoreactive neuronal cell bodies are distributed in the brain (protocerebrum, deutocerebrum, optical lobes), and at least, four pairs of neuronal cell bodies were detected in the centrally positioned neuropil of the posterior ganglion. Rich plexuses of immunoreactive nerve fibers in the neuropil area were also observed. Furthermore, bundles of strongly immunoreactive nerve fibers surrounding the gut wall were localized, and immunoreactive nerve terminals in the antennules and compound eyes were observed. These data demonstrate the presence of a serotonin-like immunoreactive substance in the barnacle cyprids; furthermore, its immunolocalization in the cephalic nerve terminals allows us to postulate the involvement of this bioactive molecule in substrate recognition during the settlement process.     

The effects of a serine protease, Alcalase, on the adhesives of barnacle cyprids (Balanus amphitrite)

Barnacles are a persistent fouling problem in the marine environment, although their effects (eg reduced fuel efficiency, increased corrosion) can be reduced through the application of antifouling or fouling-release coatings to marine structures. However, the developments of fouling-resistant coatings that are cost-effective and that are not deleterious to the marine environment are continually being sought. The incorporation of proteolytic enzymes into coatings has been suggested as one potential option. In this study, the efficacy of a commercially available serine endopeptidase, Alcalase as an antifoulant is assessed and its mode of action on barnacle cypris larvae investigated. In situ atomic force microscopy (AFM) of barnacle cyprid adhesives during exposure to Alcalase supported the hypothesis that Alcalase reduces the effectiveness of the cyprid adhesives, rather than deterring the organisms from settling. Quantitative behavioural tracking of cyprids, using Ethovision 3.1, further supported this observation. Alcalase removed cyprid 'footprint' deposits from glass surfaces within 26 min, but cyprid permanent cement became resistant to attack by Alcalase within 15 h of expression, acquiring a crystalline appearance in its cured state. It is concluded that Alcalase has antifouling potential on the basis of its effects on cyprid footprints, un-cured permanent cement and its non-toxic mode of action, providing that it can be successfully incorporated into a coating.     

Inhibitory effect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite amphitrite darwin

This study examined the inhibitory effect of 3 phenolic compounds and 12 strains of marine bacteria on the larval settlement of Balanus amphitrite amphitrite. The phenolic compounds used were phlorotannins, phloroglucinol and tannic acid. Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene) known only from brown algae. Tannic acid, which exists in terrestrial plants, is composed of oligomers of phloroglucinol attached to a sugar molecule. The bacterial strains used were isolated from a natural biofilm. The following were investigated: 1) the toxicity of the phenolic compounds to B. a. amphirite in three different larval stages, viz. nauplius II, nauplius V and cyprid; 2) the potency of the compounds as inhibitors of larval settlement and the possible mechanism involved in settlement inhibition; and 3) the effects of the bacteria on larval settlement. The level of toxicity of the phenolic compounds varied widely for the larvae. Phlorotannins were most toxic, having LC₅₀ values ranging from 9.47 to 40.35 μg ml^‐1; phloroglucinol was least toxic, having LC₅₀ values of 235.12 to 368.28 μg ml^‐1. In general, nauplii were more sensitive to the toxicity of the phenolic compounds than cyprids. The greater sensitivity of nauplii may be due to their active feeding behavior, which exposes the interior of their bodies to the compounds by active intake. Phloroglucinol was the most potent settlement inhibitor, having an EC₅₀ value of 0.02 μg ml^‐1. Phlorotannins and tannic acid had EC₅₀ values of 1.90 μg ml^‐1 and 14.05 μg ml^‐1, respectively. Phloroglucinol appeared to inhibit larval settlement through a relatively non‐toxic mechanism as its LC₅₀ value was four orders of magnitude higher than its EC₅₀ value. The high potency of phloroglucinol indicates that a simple constituent of a complex natural compound can be more effective than the natural compound itself. Larval settlement bioassays with monospecies bacterial films indicated that some of the bacterial species were inhibitory to larval settlement while the others showed no effect. None of the bacterial strains in this study induced larval settlement.     

Base plate mechanics of the barnacle Balanus amphitrite (=Amphibalanus amphitrite)

The mechanical properties of barnacle base plates were measured using a punch test apparatus, with the purpose of examining the effect that the base plate flexural rigidity may have on adhesion mechanics. Base plate compliance was measured for 43 Balanus amphitrite (=Amphibalanus amphitrite) barnacles. Compliance measurements were used to determine flexural rigidity (assuming a fixed-edge circular plate approximation) and composite modulus of the base plates. The barnacles were categorized by age and cement type (hard or gummy) for statistical analyses. Barnacles that were 'hard' (> or =70% of the base plate thin, rigid cement) and 'gummy' (>30% of the base plate covered in compliant, tacky cement) showed statistically different composite moduli but did not show a difference in base plate flexural rigidity. The average flexural rigidity for all barnacles was 0.0020 Nm (SEM +/- 0.0003). Flexural rigidity and composite modulus did not differ significantly between 3-month and 14-month-old barnacles. The relatively low flexural rigidity measured for barnacles suggests that a rigid punch approximation is not sufficient to account for the contributions to adhesion mechanics due to flexing of real barnacles during release.     

Modulation of barnacle (Balanus amphitrite Darwin) cyprid settlement behavior by sulfobetaine and carboxybetaine methacrylate polymer coatings

Zwitterionic polymers such as poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA) have demonstrated impressive fouling-resistance against proteins and mammalian cells. In this paper, the effects of these surface chemistries on the settlement and behavior of an ubiquitous fouling organism, the cypris larva of the barnacle Balanus amphitrite (=Amphibalanus amphitrite), were studied in the laboratory. Conventional settlement assays and behavioral analysis of cyprids using Noldus Ethovision 3.1 demonstrated significant differences in settlement and behavior on different surfaces. Cyprids did not settle on the polySBMA or polyCBMA surfaces over the course of the assay, whereas settlement on glass occurred within expected limits. Individual components of cyprid behavior were shown to differ significantly between glass, polySBMA and polyCBMA. Cyprids also responded differently to the two zwitterionic surfaces. On polySBMA, cyprids were unwilling or unable to settle, whereas on polyCBMA cyprids did not attempt exploration and left the surface quickly. In neither case was toxicity observed. It is concluded that a zwitterionic approach to fouling-resistant surface development has considerable potential in marine applications.     

Characterization of metalloproteinase-like activities in barnacle (Balanus amphitrite) nauplii

The presence of extracellular matrix (ECM) degrading enzymes was investigated in naupliar stages of the barnacle Balanus amphitrite Darwin. The results of substrate gel-zymography and quantitative assays demonstrated that naupliar extracts contain several protease activities that are specific towards gelatin substrates; some caseinolytic activity was also detected. Substrate specificity was observed in all naupliar stages (II-VI). The gelatinolytic activities showed dependence on both Ca(2+) and Zn(2+) and inhibition by EDTA, EGTA, and 1,10-phenanthroline. Also Mg(2+) partially activated the enzymes, whereas Cd(2+), Cu(2+), Hg(2+) and Pb(2+) were inhibitory. The thermal denaturation profile was significantly different in the presence and absence of Ca(2+) and Zn(2+). Overall, the results indicate that the Ca(2+)/Zn(2+)-dependent gelatinase activities in barnacle nauplii belong to the subfamily of matrix metalloproteases. Barnacle larvae MMPs showed biochemical characteristics different from those of vertebrate MMPs but common to other gelatinases from marine invertebrates: they were unaffected by several protease inhibitors and insensitive to specific activators/inhibitors of vertebrate MMPs. The presence of MMP-like activities in different naupliar stages suggests a constitutive role for these enzymes in ECM remodeling during barnacle larvae growth and development.     

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.     

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.     

Flavone and isoflavone derivatives of terrestrial plants as larval settlement inhibitors of the barnacle Balanus amphitrite

To determine whether they could serve as non-toxic or less damaging alternative antifouling (AF) agents, 17 flavone and isoflavone derivatives were isolated from terrestrial plant extracts, purified and examined for their ability to inhibit the settlement of barnacle (Balanus amphitrite) cyprids. In larval bioassays, eight compounds showed strong anti-larval settlement activities, with EC(50) values <10 microg ml(-1). Through an analysis of the structure-activity relationship of these compounds, it was found that (1) the structural difference between flavones and isoflavones did not affect their AF activities; (2) the 5-hydroxyl group on the skeletons played a key role in AF activities; and (3) the presence of hydroxyl group or bulky group on C3 significantly reduced AF activities. A hydrolysis experiment using genistein, a typical active compound in this study, indicated that it was decomposed in the marine environment by hydrolysis reaction and that the degradation speed was significantly affected by pH. In a field AF test, genistein inhibited the attachment of B. amphitrite on panels coated with genistein-paint mixtures.     

The bioactivity of bacterial isolates in Hong Kong waters for the inhibition of barnacle (Balanus amphitrite Darwin) settlement

Three bacterial isolates (Micrococcus sp., Rhodovulum sp., and Vibrio sp.) from natural biofilms were investigated for their effects on cyprid settlement of Balanus amphitrite in laboratory bioassays. The inhibitive effect of these bacteria was clearly demonstrated by using a choice assay, in which cyprids settled preferentially on surfaces without bacterial pretreatment over those possessing a monospecies bacterial film. This result suggested that the inhibitive effect was mediated by direct larval contact with bacterial film surface rather than the perception of diffusible bacterial products. In a no-choice assay, monospecies bacterial films of different cell densities reduced cyprid settlement in a density-dependent manner. Vibrio sp. was the most potent inhibitor among the three isolates as it effectively inhibited cyprid settlement by relatively low-density films. The cells of Vibrio sp. were the smallest among the three isolates, suggesting that the correlation between bacterial cell density and cyprid settlement might not be merely due to the reduction of free-space availability. For all three isolates, films that were killed by formaldehyde or UV treatment were as potent as untreated, live films. These films remained inhibitive even in the presence of a strong promoter for cyprid settlement, namely conspecific settlement factor (SF), obtained from adult B. amphitrite. However, SF reverted the inhibitive effect of natural biofilms developed in the intertidal region.     

Quantifying the exploratory behaviour of Amphibalanus amphitrite cyprids

The behavioural response of cypris larvae from A. amphitrite (=Balanus amphitrite) exploring three model glass surfaces is quantified by close-range microscopy. Step length and step duration measurements reveal a response to both surface properties and flow. Without flow, 2-day-old cyprids took larger steps with shorter step duration on hydrophilic glass surfaces (bare and NH₂-treated) vs hydrophobic glass (CH₃-treated). These parameters suggest a more detailed, local inspection of hydrophobic surfaces and a more extensive exploration for hydrophilic surfaces. Cyprids under flow took longer steps and exhibited shorter probing times on hydrophobic glass. On hydrophilic glass, cyprids increased their step duration under flow. This active response is attributed to drag and lift forces challenging the cyprids' temporary anchoring to the substratum. Seven-day-old cyprids showed almost no discrimination between the model surfaces. Microscopic-scale observation of cyprid exploration is expected to provide new insights into interactions between cyprids and surfaces.     

The use of computer-assisted motion analysis for quantitative studies of the behaviour of barnacle (Balanus amphitrite) larvae

The effects of larval density and age on pre-settlement swimming behaviour of Balanus amphitrite cyprid larvae were studied with the aid of computer-assisted motion analysis. Swimming behaviour was monitored in individual, in groups of 10-15 and in groups of 50-100 cyprids. There was a small, but significant effect of larval density on swimming speed and no effect on two other quantitative measures: rate of change of direction and net-to-gross displacement ratio. There was also small but significant variation in swimming speed between different batches of cyprids over the course of 2 years. Swimming behaviour of individual cyprid larvae was also monitored daily for 7 days, with the larvae maintained in the cold and dark between measurements to prevent settlement and metamorphosis. There were no significant behavioural differences observed over time indicating that larvae may be held in this manner experimentally without affecting these parameters.     

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

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

Eight polymorphic microsatellite loci markers for the barnacle Balanus amphitrite (syn. Amphibalanus amphitrite) Darwin 1854

Balanus amphitrite is a widespread species of barnacle. It is frequently studied, and of great importance to the marine coatings industry due to its significant abundance as a fouling organism on commercial shipping. We isolated and characterized eight highly polymorphic microsatellite loci, to aid in the determination of population genetic structure within this species. All loci showed considerable genetic variation with the number of alleles ranging from two to 14. Expected heterozygosity ranged from 0.74 to 0.98.     

Uptake of the neurotransmitter histamine into the eyes of larvae of the barnacle (Balanus amphitrite)

The photoreceptors of adult barnacles use histamine as their neurotransmitter and take up (3)H-histamine selectively from the extracellular medium. We assayed for the uptake of (3)H-histamine into the eyes of the free-swimming (nauplius) and settling (cyprid) larval stages of Balanus amphitrite. The extracellular space of nauplii proved permeable to dyes below about 800 molecular weight (MW), indicating that (3)H-histamine (MW 111) introduced into seawater would have access to internal structures. (3)H-Histamine was taken up into nauplii by a process with a K(D) of 0.32 microM. Uptake was antagonized by chlorpromazine, which also blocks uptake of (3)H-histamine into adult photoreceptors. In autoradiographs of serial sections of nauplii and cyprids incubated in (3)H-histamine, the ocelli and compound eyes were labeled; other structures in the animal were not. No eyes or other structures were labeled with (3)H-serotonin, a related amine whose transporter commonly transports histamine as well. These experiments show that a histamine-specific transporter similar to that found in the adult is expressed in all of the eyes of barnacle larvae. In the ocelli, where photoreceptors and pigment cells may be distinguished in the light microscope, label was unexpectedly concentrated far more over the pigment cells than over the photoreceptors.