Effect of ultrasound on cyprids and juvenile barnacles

Settlement inhibition of barnacle (Amphibalanus amphitrite) cypris larvae resulting from exposure to ultrasound was measured at three frequencies (23, 63, and 102 kHz), applied at three acoustic pressure levels (9, 15, and 22 kPa) for exposure times of 30, 150, and 300?s. The lowest settlement was observed for 23?kHz, which also induced the highest cyprid mortality. Cyprid settlement following exposure to 23?kHz at 22?kPa for 30?s was reduced by a factor of two. Observing surface exploration by the cyprids revealed an altered behaviour following exposure to ultrasound: step length was increased, while step duration, walking pace, and the fraction of cyprids exploring the surface were significantly reduced with respect to control cyprids. The basal area of juvenile barnacles, metamorphosed from ultrasound-treated cyprids was initially smaller than unexposed individuals, but normalised over two weeks' growth. Thus, ultrasound exposure effectively reduced cyprid settlement, yet metamorphosed barnacles grew normally.     

Inhibition of barnacle cyprid settlement using low frequency and intensity ultrasound

Low frequency, low intensity ultrasound was demonstrated as an effective inhibitor of barnacle cyprid settlement. When the same substratum vibration amplitude (10.05 nm) and acoustic pressure (5 kPa) were applied, ultrasound at a frequency of 23?kHz significantly reduced cyprid settlement. The mechanism appeared to differ from the ultrasonic cavitation induced inhibition previously reported as no increased mortality was observed, and no change in the exploratory behaviour of cyprids was observed when they were exposed to this continuous ultrasonic irradiation regime. The application of ultrasound treatment in an intermittent mode of '5?min on and 20?min off' at 20-25?kHz and at the low intensity of 5?kPa produced the same effect as the continuous application of 23?kHz. This energy efficient approach to the use of low frequency, low intensity ultrasound may present a promising and efficient strategy regarding irradiation treatment for antifouling applications.     

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.     

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.     

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.     

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.     

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.     

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.     

Influence of oil shale on intertidal organisms: Effect of oil shale extract on settlement of the barnacle Balanus balanoides (L.)

As part of a study to investigate the effect of oil seeps on intertidal organisms, oil extracts of Blackstone oil shale from Kimmeridge on the Dorset coast were used in laboratory experiments to test their effect on the settlement of the barnacle Balanus balanoides (L.). Thin films of oil extract painted on the surface of pits in slate panels had no effect on cyprid settlement when applied up to a surface density of 2.8 g · m^?2, representing a thickness of 3.3 μm. Larger surface densities of oil stimulated cyprids to settle in far greater numbers than on unoiled panels. The maximum effect was obtained at a surface density of between 14.0 and 56.0 g · m ^?2, representing a thickness of 16.5 μm and 66.0 μm. With higher concentration of oil in the pits, stimulation to settle was reduced although cyprid settlement was still encouraged at a surface density of oil of 112g · m^?2 or 132 μm thickness.The unfractionated crude oil shale extract was a less powerful stimulus for barnacle settlement than a partially purified solution of the integumental protein arthropodin, another strong settlement inducer for barnacle cyprids.     

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。     

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.     

Spontaneous multiscale phase separation within fluorinated xerogel coatings for fouling-release surfaces

Four-component xerogel films consisting of 1 mole-% n-octadecyltrimethoxysilane (C18) and 50 mole-% tetraethoxysilane (TEOS) in combination with 1–24 mole-% tridecafluoro-1,1,2,2-tetrahydrooctyltriethoxysilane (TDF) and 25–48 mole-% n-octyltriethoxysilane (C8) and a 1:49:50 mole-% C18/TDF/TEOS were prepared. Settlement of barnacle cyprids and removal of juvenile barnacles, settlement of zoospores of the alga Ulva linza, and strength of attachment of 7-day sporelings (young plants) of Ulva were compared amongst the xerogel formulations. Several of the xerogel formulations were comparable to poly(dimethylsiloxane) elastomer with respect to removal of juvenile barnacles and removal of sporeling biomass. The 1:4:45:50 and 1:14:35:50 C18/TDF/C8/TEOS xerogels displayed some phase segregation by atomic force microscopy (AFM) pre- and post-immersion in water. Imaging reflectance infrared microscopy showed the formation of islands of alkane-rich and perfluoroalkane-rich regions in these same xerogels both pre- and post-immersion in water. Surface energies were unchanged upon immersion in water for 48 h amongst the TDF-containing xerogel coatings. AFM measurements demonstrated that surface roughness on the 1:4:45:50 and 1:14:35:50 C18/TDF/C8/TEOS xerogel coatings decreased upon immersion in water.     

Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisus

The barnacle Balanus improvisus is the major fouling macroorganism in Swedish waters and it colonizes most man‐made surfaces submerged in the sea. New or impending legislation restricts the use of traditional, hazardous antifouling coatings based on heavy metals, mainly copper and tin. This calls for the development of new non‐toxic methods that prevent barnacle settlement. In this work several adrenoceptor compounds are shown to be very efficient in preventing the settlement of cyprid larvae of B. improvisus. The settlement rate of laboratory‐reared cyprids was studied in hydrophilised polystyrene dishes containing adrenoceptor antagonists and agonists dissolved in seawater. Two of these drugs, medetomidine and clonidine, repeatedly inhibited settlement at concentrations between 1 nM and 10 nM. In the vertebrate adrenoceptor classification system, which separates pharmacological substances according to their receptor affinity, both of these substances are classified as α₂ adrenoceptor agonists. An inhibiting effect on presyn‐aptic receptors is suggested, but the localization of the receptor effect requires futher studies. Experiments also revealed that the inhibiting effect of medetomidine was reversible. Cyprids incubated with medetomidine for 20 h attached and metamorphosed into juvenile barnacles after washing and transferrence to seawater. The antagonizing compound atipamezole reversed the effect of medetomidine. This observation supports the assumption that this substance acts at the receptor level. Studies of the surface affinity of medetomidine revealed a strong tendency to accumulate in solid/ liquid phase boundaries. This ability makes it particularly attractive as a candidate for the development of a slow‐release carrier in marine coatings. Panels coated with medetomidine in an acrylate polymer and exposed in the field reduced the recruitment of B. improvisus by 96% after 4 weeks and by 70% after 8 weeks.     

Surface exploration of Amphibalanus amphitrite cyprids on microtextured surfaces

Microtopography is one of several strategies used by marine organisms to inhibit colonization by fouling organisms. While replicates of natural microtextures discourage settlement, details of larval interactions with the structured surfaces remain scarce. Close-range microscopy was used to quantify the exploration of cyprids of Amphibalanus amphitrite on cylindrical micropillars with heights of 5 and 30 μm and diameters ranging from 5 to 100 μm. While 5 μm-high structures had little impact, 30 μm-high pillars significantly influenced cyprid exploration. An observed step length decrease and step duration increase on 5 μm diameter pillars is attributed to the small dimensions of the voids excluding the cyprid's attachment disc and consequently reducing the area of adhesive contact. When exploring larger diameter pillars, cyprids preferred using the voids to form temporary attachment points. This may enhance their resistance to flow. No-choice assay settlement patterns mirrored this exploration behaviour, albeit in a pattern counter to what was predicted.     

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.     

The effect of gregariousness in cyprid settlement assays

The in vitro settlement assay using cyprids of Balanus. amphitrite is an important tool in basic and applied research. The aim of this study was to quantify the effect of gregariousness within these assays, and to determine the interaction between gregariousness and container size, and settlement promoters and inhibitors. Assays with 1, 5, 10, 15, 20, and 40 cyprids in containers with a log range of surface area to volume ratios were conducted in a fully factorial design. Assays with the same range of cyprid numbers and six concentrations of the settlement promoter 3-isobutyl-1-methylxanthine (IBMX) or the inhibitor phloroglucinol were also conducted in a fully factorial design. Percentage settlement was analysed by GLM ANOVA. Significant (p < 0.05) gregarious effects were detected at > or = 5 cyprids in a well. Surface area:volume ratio had a strong effect on cyprid settlement, but this effect could be masked by overcrowding in very small wells. Gregarious interactions between only five cyprids magnified the effect of IBMX by a factor of 10, whereas phloroglucinol had no effect without gregarious interactions. The cyprid settlement bioassay is a valuable tool for basic and applied research but must be used with care.     

Effects of temperature and salinity on the larvae of two species of rhizocephalan (Crustacea: Cirripedia)

Summary

Responses of larvae of two rhizocephalan species to changes in seawater temperature and salinity were studied under laboratory conditions. Peltogasterella gracilis parasitizes the hermit crab Pagurus pectinatus, which occurs at stable salinity and gradually changing temperature in summer. Sacculina polygenea is a parasite of the crab Hemigrapsus sanguineus, which lives in the intertidal zone in summer where salinity and temperature can fluctuate during the day. The development of both species is comprised of five naupliar stages and the cyprid stage, and it was considered successful if more than 50% of the nauplii attained the cyprid stage. P. gracilis nauplii successfully developed at 12–20°C and 30–34‰, but at 22°C successful development occurred in a narrower salinity range (32–34‰). All nauplii died both at 25°C and in 26‰. S. polygenea nauplii successfully reached the cyprid stage at higher temperatures (18–25°C) and a wider salinity range (18–34‰) than P. gracilis nauplii, but at 12°C and 16‰ larval development of S. polygenea was suppressed. Under favorable conditions, naupliar development lasted 3.5 days in P. gracilis and 2–3 days in S. polygenea. The cyprids of both rhizocephalan species demonstrated a greater resistance to temperature and salinity changes than nauplii. However, P. gracilis cyprids were active in a narrower salinity range (16–34‰), as compared to S. polygenea cyprids (8–34‰). Under favorable conditions the cyprids of both species survived for 6 to 10 days.     

The effect of ecdysterone on cyprids of Balanus eburneus Gould

Synthetic ecdysterone in concentrations from 0.02 to 250 · 10^?6 causes developmental abnormalities in the cyprids of Balanus eburneus Gould. These cyprids are unable to attach themselves to the substratum, but readily metamorphose into barnacles with recognizable adult shell structures; these remain encased within the cyprid valves, but live only for a short time. The possible involvement of an ecdysone system in the metamorphosis of cirripedes is discussed.     

How whale and dolphin barnacles attach to their hosts and the paradox of remarkably versatile attachment structures in cypris larvae

How larvae of whale and dolphin epibionts settle on their fast-swimming and migrating hosts is a puzzling question in zoology. We successfully reared the larvae of the whale and dolphin barnacle Xenobalanus globicipitis to the cyprid stage. We studied the larval developmental ecology and antennular morphology in an attempt to assess whether an epibiotic lifestyle on this extreme substratum entails any unique larval specializations. Morphological parameters were compared with five other barnacle species that also inhabit extreme substrata. We found no larval specializations to a lifestyle associated with marine mammals. The external morphology of the antennules in Xenobalanus cyprids is morphologically similar to species from strikingly different substrata. We found variation only in the structures that are in physical contact with the substratum, i.e., the third segments carrying the villi-covered attachment disc. The third segments of the Xenobalanus cyprid antennules are not spear-shaped as in the stony coral barnacles, which are here used to penetrate the live tissue of their hosts. The presence of a cyprid cement gland implies that Xenobalanus uses cement protein when attaching to its cetacean host. Naupliar instars developed outside of the mantle cavity, indicating dispersal is planktonic. Our results militate against the idea that the cyprids settle during ocean migrations of their hosts. We suggest cyprids settle during coastal aggregations of the cetacean hosts. We conclude that the ecological success of barnacles has ultimately depended on a larva that with little structural alteration possesses the ability to settle on an amazingly wide array of substrata, including cetaceans.