Signal transduction in barnacle settlement: Calcium re-visited

The search for marine natural product antifoulants is being hampered by problems associated with conventional settlement assays. Yet it has been recognised that the study of how chemical cues are perceived by fouling organisms may offer clues to settlement inhibitors and may identify novel biochemical assays for antifoulants based on signal transduction pathways. Here the role of calcium in barnacle settlement is re-examined. A requirement for calcium in settlement of the cypris larva of Balanus amphitrite has been confirmed; settlement was inibited in low calcium, and calcium-free, seawater. Although 10 mM (19.27 mM) excess calcium had no effect and higher concentrations were inhibitory, a 5 mM excess stimulated settlement. Stimulation is proposed to be effected by an increase in intracellular calcium. The release of calcium from intracellular pools with thapsigargin (but not cyclopiazonic acid) induced settlement and an antagonist of intracellular calcium, TMB-8, generally inhibited settlement. Nevertheless, the calcium ionophore A23187 did not induce settlement at the concentrations tested. Consequently, the relative importance of external calcium and intracellular pools to increased intracellular calcium has yet to be determined. Pharmacological manipulations of calcium channels with organic and inorganic channel blockers strongly indicate calcium channel involvement in barnacle settlement. The data are summarised in an hypothetical scheme for signal transduction at settlement and are compared to those obtained for other marine invertebrate larvae.     

Involvement of reactive oxygen species in the electrochemical inhibition of barnacle (Amphibalanus amphitrite) settlement

The role of reactive oxygen species (ROS) in electrochemical biofouling inhibition was investigated using a series of abiotic tests and settlement experiments with larvae of the barnacle Amphibalanus amphitrite, a cosmopolitan fouler. Larval settlement, a measure of biofouling potential, was reduced from 43% ± 14% to 5% ± 6% upon the application of pulsed electric signals. The application of ROS scavengers such as glutathione and catalase counteracted the inhibitory effects of the electric signals, allowing settlement, and thus indicating that ROS are antifouling agents. Based on the experimental evidence, the proposed mechanism for ROS-based fouling prevention with interdigitated electrodes involved the electrochemical generation of hydrogen peroxide by oxygen reduction, and its likely reduction to hydroxyl radicals. Either hydroxyl radicals or products of hydroxyl radical reactions appeared to be the main deterrents of larval settlement.     

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.     

Expression of calmodulin and myosin light chain kinase during larval settlement of the Barnacle Balanus amphitrite

Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca(2+)/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite.     

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.     

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.     

MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.     

Bioassays and field immersion tests: a comparison of the antifouling activity of copper-free poly(methacrylic)-based coatings containing tertiary amines and ammonium salt groups

This paper focuses on the activity spectrum of three dimethylalkyl tertiary amines as potential active molecules and the corresponding ammonium salt-based antifouling (AF) paints. Bioassays (using marine bacteria, microalgae and barnacles) and field tests were combined to assess the AF activity of coatings. Bioassay results demonstrated that the ammonium salt-based paints did not inhibit the growth of microorganisms (except the dimethyldodecylammonium-based coatings) and that the tertiary amines were potent towards bacteria, diatoms, and barnacle larvae at non-toxic concentrations (therapeutic ratio, LC₅₀/EC₅₀, < 1). The results from field tests indicated that the ammonium salt-based coatings inhibited the settlement of macrofouling and the dimethylhexadecylammonium-based coatings provided protection against slime in comparison with PVC blank panels. Thus, results from laboratory assays did not fully concur with the AF activity of the paints in the field trial.     

Effect of cyprid age on the settlement of balanus amphitrite darwin in response to natural biofilms

Larval settlement of the barnacle Balanus amphitrite Darwin (Cirripedia, Balanidae) is influenced by natural biofilms. In previous work by others, discriminatory settlement of aged cyprids has been observed in response to biofilms of different age. This study extends prior work by considering the effect of the age of cyprids on the outcome of settlement assays. Settlement was investigated with 0‐day‐old (newly metamorphosed) and 5‐day‐old cyprids. Biofilms under investigation were developed in the field for periods of 5 d and 1 month, and were subsequently included in laboratory settlement assays with a choice between a filmed and an unfilmed substratum. The bioassay was modified from the conventional horizontal dish design in order to generate a low water surface‐to‐volume ratio, which served to suppress larval entrapment in an organic layer on the water surface. Irrespective of cyprid age, a clear discrimination between a filmed and an unfilmed substrata was observed, and the preference for filmed or unfilmed substratum was dependent on the age of the cyprids. Settlement of 0‐day‐old cyprids was inhibited by a biofilmed substratum whereas induction occurred with aged cyprids. This pattern of settlement was independent of biofilm age. Bacterial abundance on unfilmed substrata in treatments and controls was significantly lower than that on biofilmed surfaces, confirming that bacterial contamination did not change the qualitative option during the assay.     

Cochliomycin A inhibits the larval settlement of Amphibalanus amphitrite by activating the NO/cGMP pathway

Cochliomycin A is a compound with anti-barnacle settlement activity and low toxicity, but the molecular mechanism of the compound is unknown. Here, isobaric tags for the relative or absolute quantitation (iTRAQ) labeling proteomic method were applied to analyze changes in the proteome of Amphibalanus（=Balanus） amphitrite cyprids in response to cochliomycin A treatment. Cochliomycin A affected the cytochrome P450, glutathione S-transferase (GST) and NO/cGMP pathways, among which the NO/cGMP pathway was considered to play a key role in barnacle larval settlement, while the cytochrome P450 and the GST pathways are mainly for detoxification. The results of real-time PCR further suggested the NO/cGMP pathway was activated in response to cochliomycin A. Larval settlement assays revealed that S-methylisothiourea sulfate (SMIS) and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) rescued cyprids from cochliomycin A-induced inhibition of larval settlement. The findings supported the hypothesis that cochliomycin A inhibited barnacle larval settlement by stimulating the NO/cGMP pathway.     

Adult Prey Neutralizes Predator Nonconsumptive Limitation of Prey Recruitment

Recent studies have shown that predator chemical cues can limit prey demographic rates such as recruitment. For instance, barnacle pelagic larvae reduce settlement where predatory dogwhelk cues are detected, thereby limiting benthic recruitment. However, adult barnacles attract conspecific larvae through chemical and visual cues, aiding larvae to find suitable habitat for development. Thus, we tested the hypothesis that the presence of adult barnacles (Semibalanus balanoides) can neutralize dogwhelk (Nucella lapillus) nonconsumptive effects on barnacle recruitment. We did a field experiment in Atlantic Canada during the 2012 and 2013 barnacle recruitment seasons (May–June). We manipulated the presence of dogwhelks (without allowing them to physically contact barnacles) and adult barnacles in cages established in rocky intertidal habitats. At the end of both recruitment seasons, we measured barnacle recruit density on tiles kept inside the cages. Without adult barnacles, the nearby presence of dogwhelks limited barnacle recruitment by 51%. However, the presence of adult barnacles increased barnacle recruitment by 44% and neutralized dogwhelk nonconsumptive effects on barnacle recruitment, as recruit density was unaffected by dogwhelk presence. For species from several invertebrate phyla, benthic adult organisms attract conspecific pelagic larvae. Thus, adult prey might commonly constitute a key factor preventing negative predator nonconsumptive effects on prey recruitment.     

INSECTICIDES FOR PREVENTING BARNACLE SETTLEMENT

During the summer of 1950 experiments were made to test the use of insecticides for preventing the fouling of oyster spat collectors by the barnacle Elminius modestus.
The insecticides used were DDT, BHC (benzene hexachloride-γ-isomer) and dieldrin (hexachloro-epoxy-octahydro-dimethano-naphthalene), made up as 5% solutions and tested on egg-box collectors and quarry tiles. The tiles were used for frequent observations on settlement.
When the solutions were sprayed on to the surfaces, in sufficient quantity to give a covering of 200 mg./ft.² of active insecticide, none of them interfered with either barnacle or oyster settlement.
When the test surfaces were dipped in the solutions, DDT and BHC prevented barnacle settlement almost completely, while dieldrin reduced it by approximately 50%. Settlement of Ostrea edulis was also reduced on the treated surfaces.
Urea-formaldehyde resins made up as varnishes, to which DDT or BHC had been added, were also tested. A varnish incorporating 20% by weight of DDT provided an effective anti-fouling compound which lasted at least 3 months and completely prevented barnacle settlement.
The possibility of insecticides dissolving into the water and inhibiting barnacle growth on adjacent surfaces is discussed.     

The effect of vibration frequency and amplitude on biofouling deterrence

The use of vibration is proposed as a means of controlling the settlement of marine fouling organisms. In this study, panels with embedded lead zirconate titanate, known as PZT, were placed in the field over 3?months. The panels were vibrated at different velocity levels at frequencies between 70 and 445?Hz. It was found that barnacles (Amphibalanus variegatus Darwin and Elminius sp.) were the only fouling organisms affected by the applied vibration, and these organisms settled in significantly lower numbers when the plates were excited at specific frequencies and amplitudes. Panels vibrating at relatively higher frequencies, greater than 260?Hz, exhibited reduced barnacle settlement, whilst lower frequencies in the 70–100?Hz range had little or no effect. The settlement of other fouling organisms such as tubeworms, bryozoans, ascidians and algae did not appear to be affected by the applied excitation. The experimental results showed that increasing the velocity amplitude of vibration was a contributing factor in inhibiting barnacle settlement.     

Interaction of conspecific cues in Balanus amphitrite Darwin (Cirripedia) settlement assays: continued argument for the single-larva assay

Gregariousness in marine invertebrate larvae is an important regulator of benthic community structure. Previous laboratory settlement assays employing Balanus amphitrite Darwin cyprids found gregarious effects with as few as 3 larvae well(-1), together with modulation of such effects by chemical cues. Here, the relationship between settlement rate and larval density was rigorously tested through a fully randomised design. Seawater conditioned with adult B.amphitrite was tested alongside unconditioned seawater to determine the effect of a conspecific cue on gregarious interactions. Gregarious effects were detected in both conditioned and unconditioned seawater at < or =4 larvae well(-1). In untreated seawater, settlement rate increased linearly with larval density, levelling off at densities of > or =10 larvae well(-1). In conditioned seawater, settlement induction was observed at < or =4 larvae well(-1), switching to inhibition at 6, 8 and 10 larvae well(-1), before asymptoting at the highest densities tested. These results advocate the use of individual larvae in laboratory assays that investigate factors stimulating barnacle settlement.     

Effects of localised, low-voltage pulsed electric fields on the development and inhibition of Pseudomonas aeruginosa biofilms

This work describes the use of low-voltage (0.5 - 5 V) pulsed electric fields to prevent Pseudomonas aeruginosa biofilm development. Interdigitated electrodes (IDEs) with 29-mum spacing between 22-mum-wide electrodes, were used as a platform where the effect of localised, high-strength electric fields could be tested. Alternating current, square-wave pulses were applied to the IDEs in 1 sec intervals. A two-level, three-variable factorial design experiment was used to detect the effects of applied voltage, frequency, and pulse duty ratio (i.e. percentage of pulsing time over one cycle) on the inhibition of biofilm formation. The observations indicated that a pulse configuration of 1% duty ratio, 5 V, and 200 Hz frequency reduced the area of the electrodes covered by biofilm by 50%. In general, the application of low-duty ratio pulses had a positive effect on preventing biofouling. Comparatively, frequency and applied voltage were observed to have less influence on biofouling.     

Species specificity of barnacle settlement-inducing proteins

We previously isolated a larval settlement-inducing protein complex (SIPC) from adult extracts of the barnacle, Balanus amphitrite using a nitrocellulose membrane settlement assay. In the present study, we found that the extracts of other adult barnacles, Megabalanus rosa and Balanus eburneus, also induced the settlement of B. amphitrite cyprids although the inductive activity was slightly lower than that of conspecific extracts. Furthermore, we examined reactivity to anti-SIPC antibody in adult extracts from six species of Japanese barnacles other than B. amphitrite, brine shrimp and eight marine sessile organisms besides barnacles. The results showed that all barnacles examined contained SIPC-like proteins with slightly different molecular weight, while the other animals did not react to the antibody by immunoblot analysis. These findings suggest that species specificity in settlement-inducing proteins of barnacles is not so strict, but these proteins are characteristic to barnacle species.     

Fluctuations in settlement and survival of a barnacle Semibalanus balanoides in the White Sea intertidal

We tested whether the patchiness of barnacle settlement was determined by spatial (local) differences in settling and mortality rates. During the period of 2004–2011, annual photo-registration of a barnacles Semibalanus balanoides population was conducted on six flat rocks in the White Sea intertidal zone (Arctic). The average adult population density was 48 ind dm^?2, and the average barnacle age was 3–4 years. According to age structure, during the first year, the number of recruits dropped by three to fourfold; while in the following 2 years, the population density remained nearly the same. The effect of ice friction influence was not recorded, although the rocks were covered with ice in winter. Settling peaks were recorded in 2005, 2007, and 2009. No correlation was observed between the recruit and adult distribution during 2 years of intense settlement. The death rate was uneven on different quadrates along the transects but did not correlate with population density. Because high settling rates and mortality are not linked to specific sites, this suggests that population patchiness is not spatially stable.     

Factors influencing initial larval settlement: temporal, spatial and surface molecular components

The impact of initial surface chemistry on settlement of barnacle, bryozoan, and hydroid larvae was assessed. Temporal (on a scale of weeks to months) and fine scale spatial (centimeter to meter) variation in settlement were quantified. Four arrays of silanized glass surfaces, deployed at ≈2-wk intervals, were monitored after 1 and 3 days of immersion. Settlement of all larval types exhibited strong temporal variation. There was a 25, 22- and 18-fold difference between the highest and lowest Day 1 settlement for barnacles, bryozoans and hydroids, respectively. Bryozoan and hydroid settlement was spatially variable, barnacle settlement was not. Barnacle and bryozoan settlement was influenced by inttial surface chemistry, hydroid settlement was not. For barnacles, there was a 2-fold difference between total settlement over the four arrays on untreated glass and diphenyl-silanized surfaces; for bryozoans, there was a 51-fold difference, and in the opposite direction. There was a negative correlation between barnacle and bryozoan settlement with respect to surface. This result was independent of the presence of the other species. Bryozoan settlement was also spatially and temporally quantified in a separate, single surface 56-h array, and total bryozoan settlement ·h⁻¹ was found to be correlated with light intensity. Patterns of initial colonization were strongly influenced by surface chemistry and spatial temporal variation in larval supply. These effects on initial colonization may influence subsequent community development.     

Larval development and settlement of a whale barnacle

Larval development and settlement of whale barnacles have not previously been described, unlike intertidal barnacles. Indeed, the mechanisms of the association between barnacles and whales have not been studied. Here we describe the larval development and settlement of the whale barnacle, Coronula diadema, and possible involvement of a cue from the host in inducing larval settlement. Eight-cell stage embryos were collected from C. diadema on a stranded humpback whale, incubated in filtered seawater for 7 days, and nauplius larvae hatched out. When fed with Chaetoceros gracilis, the nauplii developed to stage VI, and finally metamorphosed to the cypris stage. The larval development looked similar to that of intertidal barnacles with planktotrophic larval stages. The cyprids did not settle in normal seawater, but did settle in polystyrene Petri dishes when incubated in seawater with a small piece of skin tissue from the host whale. This strongly suggests the involvement of a chemical cue from the host whale tissue to induce larval settlement.     

Volatile substances from adult extracts induce larval settlement of the barnacle balanus amphitrite *

The volatile substance extracted from conspecific adults induces larval settlement of the barnacle Balanus amphitrite. The settlement inducing activity of the volatile fractions was checked monthly from June, 1997 to December, 1998. Both water soluble extracts and volatile fractions from the barnacle were prepared by the steam distillation. The active cue in the volatile fraction was always extracted with n‐pentane under acidic conditions, although settlement inducing activity varied with the sample. GC‐MS analysis of the active and inactive pentane fractions revealed 1, 2, 3‐trimethyl‐benzene as the settlement inducing substance. Commercially available 1, 2, 3‐trimethylbenzene also showed high settlement inducing activity at a concentration of 0.8 × 10^‐12 M (100 pg 1^‐1). This substance was detected at concentrations of more than 7 ng g^‐1 of wet barnacle (equivalent to 0.6 × 10^‐12M, equal to 70pg1^‐1) by GC analysis. These results indicate that 1,2,3‐trimethylbenzene in the volatile fractions acts as a chemical cue for larval settlement. Monthly variation in the settlement inducing activity was observed, which synchronized with the breeding season of the barnacle. This observation suggests that the barnacle produced the chemical cue in the gonad during maturation or accumulated it from the environment.