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.     

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%.     

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.     

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.     

Presence and distribution of serotonin immunoreactivity in the cyprids of the barnacle Balanus amphitrite

In this work, the presence and distribution of serotonin in the cyprid of the barnacle Balanus amphitrite were investigated by immunohistochemical methods. Serotonin-like immuno-reactive neuronal cell bodies were detected in the central nervous system only. Various clusters of immunoreactive neuronal cell bodies are distributed in the brain (protocerebrum, deutocerebrum, optical lobes), and at least, four pairs of neuronal cell bodies were detected in the centrally positioned neuropil of the posterior ganglion. Rich plexuses of immunoreactive nerve fibers in the neuropil area were also observed. Furthermore, bundles of strongly immunoreactive nerve fibers surrounding the gut wall were localized, and immunoreactive nerve terminals in the antennules and compound eyes were observed. These data demonstrate the presence of a serotonin-like immunoreactive substance in the barnacle cyprids; furthermore, its immunolocalization in the cephalic nerve terminals allows us to postulate the involvement of this bioactive molecule in substrate recognition during the settlement process.     

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.     

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.     

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.     

Inhibitory effect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite amphitrite darwin

This study examined the inhibitory effect of 3 phenolic compounds and 12 strains of marine bacteria on the larval settlement of Balanus amphitrite amphitrite. The phenolic compounds used were phlorotannins, phloroglucinol and tannic acid. Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene) known only from brown algae. Tannic acid, which exists in terrestrial plants, is composed of oligomers of phloroglucinol attached to a sugar molecule. The bacterial strains used were isolated from a natural biofilm. The following were investigated: 1) the toxicity of the phenolic compounds to B. a. amphirite in three different larval stages, viz. nauplius II, nauplius V and cyprid; 2) the potency of the compounds as inhibitors of larval settlement and the possible mechanism involved in settlement inhibition; and 3) the effects of the bacteria on larval settlement. The level of toxicity of the phenolic compounds varied widely for the larvae. Phlorotannins were most toxic, having LC₅₀ values ranging from 9.47 to 40.35 μg ml^‐1; phloroglucinol was least toxic, having LC₅₀ values of 235.12 to 368.28 μg ml^‐1. In general, nauplii were more sensitive to the toxicity of the phenolic compounds than cyprids. The greater sensitivity of nauplii may be due to their active feeding behavior, which exposes the interior of their bodies to the compounds by active intake. Phloroglucinol was the most potent settlement inhibitor, having an EC₅₀ value of 0.02 μg ml^‐1. Phlorotannins and tannic acid had EC₅₀ values of 1.90 μg ml^‐1 and 14.05 μg ml^‐1, respectively. Phloroglucinol appeared to inhibit larval settlement through a relatively non‐toxic mechanism as its LC₅₀ value was four orders of magnitude higher than its EC₅₀ value. The high potency of phloroglucinol indicates that a simple constituent of a complex natural compound can be more effective than the natural compound itself. Larval settlement bioassays with monospecies bacterial films indicated that some of the bacterial species were inhibitory to larval settlement while the others showed no effect. None of the bacterial strains in this study induced larval settlement.     

Molecular cloning of a putative serotonin receptor gene from barnacle,Balanus amphitrite

We isolated a putative serotonin receptor gene from a genomic library of the barnacle, Balanus amphitrite Darwin, using an NcoI fragment of the barnacle G protein-coupled receptor gene that is homologous to the α₂-adrenoceptor. The cloned genomic DNA had no intron and specified an open reading frame of 1137 base pairs encoding 379 amino acids (aa). The predicted aa sequence has a typical seven hydrophobic transmembrane spanning region and a consensus G protein-binding motif. This receptor was most homologous to the human 5HT_1A receptor and closely related to other 5HT₁ receptor subtypes.     

In situ ATR–FTIR characterization of primary cement interfaces of the barnacle Balanus amphitrite

A method is presented for characterizing primary cement interfaces of barnacles using in situ attenuated total reflection–Fourier transform infrared spectroscopy. Primary cement of the barnacle, Balanus amphitrite (Amphibalanus amphitrite), was characterized without any disruption to the original cement interface, after settling and growing barnacles directly on double sided polished germanium wafers. High-quality IR spectra were acquired of live barnacle cement interfaces, providing a spectroscopic fingerprint of cured primary cement in vivo with the barnacle adhered to the substratum. Additional spectra were also acquired of intact cement interfaces for which the upper portion of the barnacle had been removed leaving only the base plate and cement layer attached to the substratum. This allowed further characterization of primary cement interfaces that were dried or placed in D₂O. The resulting spectra were consistent with the cement being proteinaceous, and allowed analysis of the protein secondary structure and water content in the cement layer. The estimated secondary structure composition was primarily β-sheet, with additional α-helix, turn and unordered components. The cement of live barnacles, freshly removed from seawater, was estimated to have a water content of 20–50% by weight. These results provide new insights into the chemical properties of the undisturbed barnacle adhesive interface.     

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.     

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.     

Flavone and isoflavone derivatives of terrestrial plants as larval settlement inhibitors of the barnacle Balanus amphitrite

To determine whether they could serve as non-toxic or less damaging alternative antifouling (AF) agents, 17 flavone and isoflavone derivatives were isolated from terrestrial plant extracts, purified and examined for their ability to inhibit the settlement of barnacle (Balanus amphitrite) cyprids. In larval bioassays, eight compounds showed strong anti-larval settlement activities, with EC(50) values <10 microg ml(-1). Through an analysis of the structure-activity relationship of these compounds, it was found that (1) the structural difference between flavones and isoflavones did not affect their AF activities; (2) the 5-hydroxyl group on the skeletons played a key role in AF activities; and (3) the presence of hydroxyl group or bulky group on C3 significantly reduced AF activities. A hydrolysis experiment using genistein, a typical active compound in this study, indicated that it was decomposed in the marine environment by hydrolysis reaction and that the degradation speed was significantly affected by pH. In a field AF test, genistein inhibited the attachment of B. amphitrite on panels coated with genistein-paint mixtures.     

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．     

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 bioactivity of bacterial isolates in Hong Kong waters for the inhibition of barnacle (Balanus amphitrite Darwin) settlement

Three bacterial isolates (Micrococcus sp., Rhodovulum sp., and Vibrio sp.) from natural biofilms were investigated for their effects on cyprid settlement of Balanus amphitrite in laboratory bioassays. The inhibitive effect of these bacteria was clearly demonstrated by using a choice assay, in which cyprids settled preferentially on surfaces without bacterial pretreatment over those possessing a monospecies bacterial film. This result suggested that the inhibitive effect was mediated by direct larval contact with bacterial film surface rather than the perception of diffusible bacterial products. In a no-choice assay, monospecies bacterial films of different cell densities reduced cyprid settlement in a density-dependent manner. Vibrio sp. was the most potent inhibitor among the three isolates as it effectively inhibited cyprid settlement by relatively low-density films. The cells of Vibrio sp. were the smallest among the three isolates, suggesting that the correlation between bacterial cell density and cyprid settlement might not be merely due to the reduction of free-space availability. For all three isolates, films that were killed by formaldehyde or UV treatment were as potent as untreated, live films. These films remained inhibitive even in the presence of a strong promoter for cyprid settlement, namely conspecific settlement factor (SF), obtained from adult B. amphitrite. However, SF reverted the inhibitive effect of natural biofilms developed in the intertidal region.     

Locating the barnacle settlement pheromone: spatial and ontogenetic expression of the settlement-inducing protein complex of Balanus amphitrite

Barnacles are prominent members of hard substratum benthic communities and their study has been important to advances in experimental ecology and contemporary ecological theory. Having recently characterized the cue to gregarious settlement of Balanus amphitrite, the settlement-inducing protein complex (SIPC), we use two polyclonal antibodies to examine the tissue distribution and ontogenetic expression of this glycoprotein. These antibodies were raised against two separate peptides located near the N- and C-termini of the SIPC and were used to detect the glycoprotein by western blotting and immunohistochemistry. By in situ hybridization we also show that the SIPC mRNA co-occurs with the expressed glycoprotein in the cuticles of both nauplius and cypris larval stages and the adult. In the larvae, the SIPC is expressed most strongly in the mouthparts and the hindgut of the stage 2 nauplius and in the thoracopods, antennules and bivalved carapace of the cyprid. In adult B. amphitrite, the expressed SIPC is present in protein extracts of the shell and in all organs that are lined by cuticular tissues. We suggest that the SIPC is produced by the epidermal cells that secrete the cuticle and discuss these observations with regard to earlier studies and the role of the SIPC as a contact pheromone.