Spectral Sensitivity of the Barnacle, Balanus amphitrite

The extracellular ocellar potential was used to evaluate the spectral sensitivity of the ocellus of the barnacle, Balanus amphitrite. Maximum relative sensitivity was at 530–540 nm. Studies with chromatic adapting lights suggest that the receptors contain a single photopigment. The spectra were relatively broader in the dark as compared to the light-adapted state. This effect was shown to be due to an increase in the slope of the amplitude-intensity function, caused by light adaptation. Studies of tapetal fluorescence and corneal transmission indicate little effect of the ocellar media on the determination of sensitivity.     

Indole Derivatives as Potent Inhibitors of Larval Settlement by the Barnacle,Balanus amphitrite

A potent inhibitor of larval settlement by the barnacle, Balanus amphitrite, was isolated as 2,5,6-tribromo-1-methylgraimne from a marine invertebrate. In comparative tests on the activity of related compounds, such compounds as 2-methylgramine and 2-methyl-3-(morpholinomethyl)-indole exhibited potent inhibitory activity. The inhibitory activity toward larval settlement was found not to be due to toxicity but to a repellent effect.     

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.     

Inhibitory Effects of Mediterranean Sponge Extracts and Metabolites on Larval Settlement of the Barnacle Balanus amphitrite

One of the most promising alternative technologies to antifouling paints based on heavy metals is the development of coatings whose active ingredients are compounds naturally occurring in marine organisms. This approach is based on the problem of epibiosis faced by all marine organisms and the fact that a great number of them cope with it successfully. The present study investigated the antifouling activity of a series of extracts and secondary metabolites from the epibiont-free Mediterranean sponges Ircinia oros, I. spinosula, Cacospongia scalaris, Dysidea sp., and Hippospongia communis. Antifouling efficacy was evaluated by the settlement inhibition of laboratory-reared Balanus amphitrite Darwin cyprids. The most promising activity was exhibited by the metabolites 2-[24-acetoxy]-octaprenyl-1-4-hydroquinone (8a), dihydrofurospongin II (10), and the alcoholic extract of Dysidea sp.     

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.     

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.     

Spatial pattern in a low-density population of the barnacle Balanus amphitrite Darwin

The spatial distribution of an intertidal population of the barnacleBalanus amphitrite was studied close to a sewage outfall nearQuequén, Argentina (38° 34S,58° 38 30 W). All individuals within an areaof 0.8 m² were mapped during 12 censuses, from September 1990to May 1993. Population density within the study area varied between 401 and99 ind m^-2. Spatial pattern was analysed using mean distancesto the nearest neighbour (NND), goodness of fit tests between observed andexpected frequency distributions of NND, and analysis of crowding. Thespatial pattern of the population was clumped during most of the studyperiod. This trend persisted, although not reaching statisticalsignificance, when the population density decreased as a consequence ofmortality and failure of successive annual recruitments. The cohortrecruited in summer 1992 (49 individuals) was randomly distributed relativeto adult barnacles already present within the study area. No consistentrelationship was observed between mortality and NND.     

Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.     

Roles of dopamine and serotonin in larval attachment of the barnacle, Balanus amphitrite

In order to clarify the roles of neurotransmitters including serotonin and dopamine in larval settlement (attachment) and metamorphosis of the barnacle Balanus amphitrite, the effects of lisuride, which acts as both a serotonin agonist/antagonist and a dopamine agonist, were examined. Lisuride did not induce larval attachment and metamorphosis; however, it promoted only larval behavior of searching for attachment sites without actual attachment to substrata which lasted for 5 to 6 days in a dose-dependent manner. Further evidence was obtained with a range of agonists/antagonists; serotonin agonists promoted the attachment, while serotonin antagonists inhibited it. Similarly, dopamine agonists inhibited the attachment. Furthermore, mixtures of serotonin and dopamine showed similar effects to those of lisuride. These results suggested that the promotion effect on larval searching behavior was derived from a combination of activities of serotonin and dopamine. Moreover, both serotonin and dopamine were detected in cyprids by HPLC. Thus, larval attachment process is regulated by both serotonin and dopamine neurons in this species. J. Exp. Zool. 284:746-758, 1999. Copyright 1999 Wiley-Liss, Inc.     

Relevance of biofilm bacteria in modulating the larval metamorphosis of Balanus amphitrite

Natural microbial communities found on different substrata exposed to the marine environment, including barnacle shell surfaces, are reported to have varying influences on the settlement and metamorphosis of competent cypris larvae. Experiments were carried out to compare the influence of settlement-inducing compounds from the bacteria isolated from the shell surface of Balanus amphitrite on its larval metamorphosis. The effect of multispecies bacterial film was also assessed. The production of different molecules by the bacteria was influenced by the nutrient media under which they were grown. It was observed that the promotory multispecies bacterial film turned to inhibition mode in the presence of the adult extract of the barnacle, indicating that bacteria-adult extract interactions alter the synthesis of different compounds produced by bacteria. The studies also show that the waterborne and the surface-associated cues from the bacteria function differentially in mediating larval metamorphosis. Understanding the complexities involved in such interactions and identification of the factors governing them would be a step forward.     

Variation in toxicity of copper pyrithione among populations and families of the barnacle,Balanus amphitrite

Inter- and intra-population variation in the toxicity of the antifouling biocide copper pyrithione (CuPT) was examined for nauplius larvae of the barnacle Balanus amphitrite. Nauplii were collected from brooding adults from four sites within the Newport River estuary (NC), chosen based on an initial estimation of recent and historical human activities that affect local contamination levels. Each site was characterized for the presence of polycyclic aromatic hydrocarbons and for the frequency of gastropod imposex, an indicator of contamination by organotins. Sensitivity of nauplii to CuPT varied significantly across the sites/populations, with LC₅₀ values ranging from 4.0 μg l^?1 to 6.1 μg l^?1. Larvae from the most contaminated site were the most sensitive to CuPT. Intrapopulation variation in toxicity was investigated by exposing nauplius larvae from 15 maternal families to a fixed concentration of CuPT (6.1 μg l^?1). Variation in larval mortality among the families was significant, ranging from 15.1% to 98.9%.     

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

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

Variation among families for characteristics of the adhesive plaque in the barnacle Balanus amphitrite

A quantitative genetics approach was used to examine variation in the characteristics of the adhesive plaque of the barnacle Balanus amphitrite Darwin attached to two silicone substrata. Barnacles settled on silicone polymer films occasionally form thick, soft adhesive plaques, in contrast to the thin, hard plaques characteristic of attachment to other surfaces. The proportion of barnacles producing a thick adhesive plaque was 0.31 for Veridian, a commercially available silicone fouling-release coating, and 0.18 for Silastic T-2, a silicone rubber used for mold-making. For both materials, significant variation among maternal families in the proportion of barnacles producing a thick adhesive plaque was observed, which suggests the presence of genetic variation, or maternal environmental effects, for this plaque characteristic. For the Veridian coating, barnacles expressing the thick adhesive plaque also exhibited significantly reduced tenacity. This represents the first reported case for potential genetic control of intraspecific phenotypic variation in the physical characteristics and tenacity of the adhesive of a fouling invertebrate.     

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.     

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 VIth 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 IInd 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 IVth stage nauplii was less than that for IInd 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.     

Boschmaella japonica,New Species,a Parasite on the Barnacles Chthamalus challengeri and Balanus amphitrite amphitrite from Japan (Crustacea: Cirripedia: Rhizocephala: Chthamalophilidae)

Abstract A new rhizocephalan parasite which infests the barnacles Chthamalus challengeri and Balanus amphitrite amphitrite is described. It is known only from the type locality, Aburatsubo, Kanagawa Prefecture, Japan. Its position in the family Chthamalophilidae is assured by the cyprids lacking a thorax, by the position of the mesenteric canal opposite to the stalk, and by its host being a balanomorph barnacle. Among the three previously described species of the Chthamalophilidae, the new species most closely resembles Boschmaella balani, but it differs in details of the internal root system and in the externa being found both on the host body and on the inner mantle. About two-thirds of the infested hosts carry more than one externa.     

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

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