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.     

Remodeling of the Nauplius Eye into the Adult Ocelli during Metamorphosis of the Barnacle, Balanus amphitrite hawaiiensis

The planktonic barnacle larva has a single median ocellus (nauplius eye), while the adult possesses two distinct sets of photoreceptors; a pair of lateral ocelli and a single median ocellus. The nauplius eye of the cypris larva of Balanus amphitrite hawaiiensis is composed of 14 visual cells grouped into three components (a pair of lateral components and a single ventral component) surrounding two centrally located pigment cells; each lateral component consists of 5 visual cells and the ventral component, 4 visual cells. In each component, the rhabdom is made up of apposing microvilli arising directly from the neighboring visual cell bodies.
During metamorphosis into the adult form, the three components of the median ocellus become separated. Each lateral component migrates laterally on the mantle and is remodeled into the adult lateral ocellus, losing two visual cells but gaining new pigment and tapetum cells in the process. The ventral component remains in the mid portion and becomes the adult median ocellus without fundamental modification in composition. The visual cells in both ocelli undergo a marked increase in volume and form many finger-like dendrites. Rhabdomes are made up of interdigitating microvilli arising from the the dendrite tips.     

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.     

舟山虾塘纹藤壶生态及生物学研究

对虾塘纹藤壶的繁殖、附着和生长的研究表明,其繁殖期在４月中旬至１０月间,附着期在５至１０月间,水温１８．９—２０．５℃．附着量除与水温有关外,还与虾塘位置、附着基的粗糙度、颜色和角度有关,最大附着量为３３０１３７个·ｍ－２,最大湿重为１３８６８．３３ｇ·ｍ－２．纹藤壶底径达１０．５ｍｍ前生长快,其后生长变缓．纹藤壶在舟山海域分布的限制因子,是波浪和透明度．     

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.     

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.     

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

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

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%.     

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

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

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.     

Studies on the factors influencing larval settlement in Balanus amphitrite and Mytilus galloprovincialis

Understanding of factors influencing settlement(attachment and metamorphosis) of marine invertebratelarvae is of great importance in aquaculture andcontrol of biofouling. The influence of two factors onsettlement of larvae was assessed from two separateinvestigations: 1, the influence of age (endogenousfactor) on cyprids of the barnacle Balanusamphitrite; and 2, the influence of a microbial film(exogenous factor) on pediveligers of the mussel Mytilus galloprovincialis.The settlement response of cypris larvae of B.amphitrite was found to be age-dependent. Oldercyprids responded more readily to settlement factorsthan newly molted ones. In M.galloprovincialis, competent pediveligers settled inresponse to a microbial filmed surface but not toan unfilmed surface. Moreover, a factor with MW of lessthan 5000 dalton, derived from culture medium of abacterial strain C1.1 (Pseudomonas-Alteromonasgroup), induced the settlement of M. galloprovincialis larvae.Thus, marine invertebrate larvae may require a periodof competence acquisition, during which they arepoorly responsive to settlement inducers. Uponacquisition of competence, larvae readily respondto external cues (e.g. microbial film, bacterialextracellular products).     

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.