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.     

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.     

Discrimination at settlement in barnacles: Laboratory and field experiments on settlement behaviour in response to settlement‐inducing protein complexes

A major driving force to mechanistic studies of barnacle gregarious settlement is to contribute to an understanding of observed patterns of settlement in nature. In particular, how cyprids perceive adult conspecifics and how they discriminate between conspecific and allospecific barnacles are questions which have taxed researchers for nearly 50 years. The putative, active component of adult barnacles to which the cyprids respond has long been known to be a glycoprotein, referred to here as the settlement‐inducing protein complex (SIPC). The present study examines the discriminatory abilities of laboratory‐reared Balanus amphitrite and wild Semibalanus balanoides cyprids at settlement. Using a recently developed nitrocellulose membrane‐choice settlement assay, laboratory studies revealed that both species settled at a significantly higher rate on regions of membrane on which crude conspecific SIPC had been adsorbed compared to untreated regions. Moreover, when offered a choice between conspecific and allospecific SIPC, a trend to greater settlement on the conspecific regions was observed. The membrane assay was also evaluated in field trials using real‐time video footage of cyprid searching behaviour. Of 211 S. balanoides cyprids recorded during exploratory behaviour, only one settled. Exploratory behaviour was, however, clearly associated with regions of the membrane treated with either conspecific or allospecific (B. Amphitrite) SIPC compared to untreated regions. These results are generally in accord with previous reports on the discriminatory abilities of barnacle cyprids and suggest that the membrane assay may be usefully applied to field studies of settlement behaviour.     

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.     

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.     

Analysis of behavioural rejection of micro-textured surfaces and implications for recruitment by the barnacle Balanus improvisus

Experiments performed in the field and in the laboratory show that the barnacle, Balanus improvisus, preferentially settles on smooth surfaces. Settlement and recruitment of B. improvisus was evaluated on micro-textured surfaces with scales of surface texture ranging from 1 to 100 μm in profile heights. Surface texture with profile heights within a topographic range of 30-45 μm reduced settlement and recruitment by 92% as compared to smooth surfaces. The reduction in recruitment on micro-textured surfaces is best explained by behavioural responses to surface topography. Behavioural experiments show that cyprids have a higher propensity for smooth surfaces than for micro-textured surfaces. Cyprids spend more time exploring smooth surfaces and more time swimming when exposed to micro-textured surfaces. Micro-textured surfaces are more often rejected by cyprids after exploration than smooth surfaces. It is suggested that some scales of surface texture could be exploited to improve future anti-fouling techniques in geographical areas where Balanus improvisus is a severe fouling problem.     

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.     

Functional polymer brushes via surface-initiated atom transfer radical graft polymerization for combating marine biofouling

Dense and uniform polymer brush coatings were developed to combat marine biofouling. Nonionic hydrophilic, nonionic hydrophobic, cationic, anionic and zwitterionic polymer brush coatings were synthesized via surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-hydroxyethyl methacrylate, 2,3,4,5,6-pentafluorostyrene, 2-(methacryloyloxy)ethyl trimethylammonium chloride, 4-styrenesulfonic acid sodium and N,N′-dimethyl-(methylmethacryloyl ethyl) ammonium propanesulfonate, respectively. The functionalized surfaces had different efficacies in preventing adsorption of bovine serum albumin (BSA), adhesion of the Gram-negative bacterium Pseudomonas sp. NCIMB 2021 and the Gram-positive Staphylococcus aureus, and settlement of cyprids of the barnacle Amphibalanus amphitrite (=Balanus amphitrite). The nonionic hydrophilic, anionic and zwitterionic polymer brushes resisted BSA adsorption during a 2 h exposure period. The nonionic hydrophilic, cationic and zwitterionic brushes exhibited resistance to bacterial fouling (24 h exposure) and cyprid settlement (24 and 48 h incubation). The hydrophobic brushes moderately reduced protein adsorption, and bacteria and cyprid settlement. The anionic brushes were least effective in preventing attachment of bacteria and barnacle cyprids. Thus, the best approach to combat biofouling involves a combination of nonionic hydrophilic and zwitterionic polymer brush coatings on material surfaces.     

Exploratory behaviour of barnacle larvae in field conditions

The aim of this work was to develop techniques for the real time filming of Semibalanus balanoides cyprid larvae in the field in order to describe the exploratory behaviour of S. balanoides cyprids under natural field conditions. The underwater camera system consisted of a high resolution, remotely controlled colour camera attached to a cradle which could be deployed from a pier. A light utilising a battery of far‐red light‐emitting diodes (λ_min = 615 nm) was used for filming at night. Transparent acrylic treated with barnacle settlement factor and smooth green polyester tiles were used as targets. From the films the tracks of the exploring cyprids were digitised from the 30 min trials. During five, 30 min trials a total of 1014 cyprids explored the surface. The numbers of cyprids exploring the surface varied from 49 to 522 cyprids per trial, and a Monte‐Carlo randomisation model showed that larval supply was random or aggregated. The mean exploration time spent on the target was 163 s with a mean distance travelled on the target of 8.5 cm. Exploratory track length was related to water current velocity by a negative curvilinear regression (R² = 0.92, F = 45.65, p < 0.05). There were significant differences in exploratory behaviour between day and night for total distance travelled (p < 0.001), straight line distance (p < 0.001) and velocity (p < 0.001) and the mean heading angle of the exploratory track (p < 0.01). The results are discussed in relation to settlement patterns and the screening of antifouling 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.     

Probing the settlement signals of Amphibalanus amphitrite

To achieve their reproductive potential, barnacles combine tactile exploration of surface structural properties and integration of cellular signals originating from their antennular sensory setae within a developmentally defined, temporally narrow window of settlement opportunity. Behavioural assays with cyprids coupled with biometric analysis of scanning electron microscopy-acquired images in the presence of specific chemical compounds were used to investigate how settlement on a substratum is altered in response to the presence of these compounds. Impeding tactile exploration was shown which altered cellular signalling and/or induced malformation of anatomical features of the antennular sensory setae, which disrupted the settlement behaviour of the model barnacle species Amphibalanus amphitrite. It is concluded that surface exploration by the cyprids relies on mechanical and nociception-related and calcium-mediated signals while a protein kinase C signalling cascade controls the timely metamorphosis of the cyprids to sessile juveniles.     

Lethal and sub‐lethal impacts of pulsed laser irradiations on the larvae of the fouling barnacle Balanus amphitrite

Laboratory experiments were conducted to study the impact of laser irradiation on the larvae of the fouling barnacle Balanus amphitrite. Research pertaining to fouling invertebrate larvae‐laser interaction is sparse and, hence, data on this aspect were thought significant in order to consider pulsed low power laser irradiations as a possible future antifouling tool. Lethal and sub‐lethal impacts of four very low laser fluences, viz. 0.013, 0.025, 0.05 and 0.1 J cm^‐2 for three different durations, viz. 2, 10 and 30 s were investigated. Three growth stages of barnacle larvae, viz. nauplii stage II, nauplii stage IV and cyprids were exposed to the mentioned laser fluences for different durations. While lethal impact was assessed immediately after and 1 d after irradiation, sub‐lethal impacts were studied by monitoring the success rate of the irradiated nauplii in reaching the cyprid stage. In addition, the swimming speed of VI^th stage nauplii after irradiation was studied. In the case of cyprids, in addition to the mortality measurement immediately after and 1 d after irradiation, the settlement rate was investigated. In all the above experiments, non‐irradiated larvae served as controls. The results showed an increase in mortality with increasing laser fluence and duration of irradiation. Irradiation for 2 s resulted in significant mortality in nauplii, while it was less in the case of cyprids. In II^nd stage nauplii, the mortality immediately after irradiation for 2 s varied from 14.8±2.12 to 97.1±4.1% for laser fluences of 0.013 and 0.1 J cm^‐2, respectively. However, in cyprids, the mortality immediately after irradiation for 2 s varied from 12.2±3 to 13.4±1.2% for fluences of 0.013 and 0.1 J cm^‐2, respectively. The mortality in IV^th stage nauplii was less than that for II^nd stage nauplii but more than that for cyprids. There was a significant increase in mortality with time after irradiation. The formation of cyprids from the irradiated larvae was significantly less than that observed for non‐irradiated larvae. Also, the irradiated larvae showed a significantly slower swimming speed compared to the control samples. The settlement rate in cyprids was reduced significantly by the laser irradiation. This was true even for the lowest fluence and shortest period of irradiation tested. Thus, the results of the experiment showed that even a low power pulsed laser irradiation of 0.013 J cm^‐2 for 2 s can cause significant damage to fouling barnacle larvae.     

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.     

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.     

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) (CH(3)-, OH-, COOH-, N(CH(3))(3) (+)-, NH(2)-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 CH(3)- 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.     

Species-specific engineered antifouling topographies: correlations between the settlement of algal zoospores and barnacle cyprids

Novel, non-toxic antifouling technologies are focused on the manipulation of surface topography to deter settlement of the dispersal stages of fouling organisms. This study investigated the effect of the aspect ratio (feature height/feature width) of topographical features engineered in polydimethylsiloxane, on the settlement of cyprids of Balanus amphitrite and zoospores of Ulva linza. The correlation of relative aspect ratios to antifouling efficacy was proven to be significant. An increase in aspect ratio resulted in an increase of fouling deterrence for both zoospores and cyprids. The spore density of Ulva was reduced 42% with each unit increase in aspect ratio of the Ulva-specific Sharklet AF topography. Similarly, the number of settled cyprids was reduced 45% with each unit increase in aspect ratio. The newly described barnacle-specific Sharklet AF topography (40 microm feature height, aspect ratio of 2) reduced cyprid settled by 97%. Techniques have been developed to superimpose the smaller Ulva-specific topographies onto the barnacle-specific surfaces into a hierarchical structure to repel both organisms simultaneously. The results for spore settlement on first-generation hierarchical surfaces provide insight for the efficacious design of such structures when targeting multiple settling species.     

The relation between cypris ultrastructure and metamorphosis in male and female Sacculina carcini (Crustacea,Cirripedia)

Summary To elucidate current controversies on sex in rhizocephalan barnacles, broods of Sacculina carcini, infesting the shore crab Carcinus maenas, were raised to cyprids in the laboratory and followed through settlement and metamorphosis. Free-swimming cyprids were studied by transmission electron microscopy and occur in male and female morphological types, which differ in the structure of carapace cuticle, antennular cuticle, antennular glands, and the cells suspected of being the stem cells during metamorphosis. These dissimilarities are in addition to the already known differences in cypris size, in number of antennular sense organs, and in substrata settled on by morphological males and females. Metamorphosing males (trichogons) and females (kentrogons) are illustrated in interference phase-contrast micrographs. The morphological differences between male and female cyprids are directly related to their dissimilar metamorphosis. Hence, cyprids of male morphology are anatomically incapable of metamorphosing into kentrogons, while cyprids of female morphology cannot metamorphose into trichogons. The determination of sex in rhizocephalan barnacles is discussed.The results refute the hypothesis that sex in Sacculina carcini is determined environmentally, e.g., by the substratum encountered by the cyprids at settlement. It is concluded that sex is determined already in the free-swimming larvae and, most probably, already in the ovary. This agrees with the mode of sex determination in other species of the Rhizocephala Kentrogonida.     

Field-based video observations of wild barnacle cyprid behaviour in response to textural and chemical settlement cues

Many marine invertebrate larvae respond behaviourally to environmental settlement cues, yet behaviours are often only inferred from settlement patterns or are limited to laboratory studies. The behaviour of wild cypris larvae of Semibalanus balanoides L. was filmed on settlement tiles in the field. Tiles were of five different textures with a nested treatment of crude conspecific adult extract (AE). The effects of texture and AE on eleven defined behaviours were analysed. Texture affected the gross and net exploratory distances, velocity, acceleration and time spent exploring. AE attracted more cyprids during the first minute of immersion and increased the time spent on surfaces. Relatively few arrivals that either travel far and fast, or exit the surface rapidly, may indicate a lower chance of settlement. An increase in time spent on a surface may increase the probability of being in contact with the surface when the sign stimulus to settle occurs.     

藤壶金星幼虫附着变态机制

藤壶属节肢动物门(Arthropoda)甲壳亚门(Crustacea)蔓足下纲(Cirripedia)围胸总目(Thoracica), 具备特殊的形态结构、生活史和种群生态特征,是最主要的海洋污损生物。其幼虫阶段通常经历6期无节幼体和1期不摄食的金星幼虫,从浮游的金星幼虫附着变态成固着的稚体是藤壶生活史中的一个关键环节。外界化学和生物因子中成体提取物、水溶性信息素、足迹、神经递质、激素、生物膜等均影响藤壶金星幼虫的附着变态;内在因子即金星幼虫的生理状态(能量储量和年龄)决定了其对外界因子的反应程度。概括了近年来藤壶附着变态生理机制和分子机制研究的进展,可为深入了解藤壶金星幼虫附着变态机制提供参考,也为开发新型、高效、环保的防污剂提供理论指导。