Evidence for different pharmacological targets for imidazoline compounds inhibiting settlement of the barnacle Balanus improvisus

We describe the effect of eight different imidazoline/guanidinium compounds on the settlement and metamorphosis of larvae of the barnacle Balanus improvisus. These agents were chosen on the basis of their similar pharmacological classification in vertebrates and their chemical similarity to medetomidine and clonidine, previously described as highly potent settlement inhibitors (nanomolar range). Seven of the tested compounds were found to inhibit settlement in a dose-dependent manner in concentrations ranging from 100 nM to 10 microM without any significant lethal effects. In vertebrate systems these substances have overlapping functions and interact with both alpha-adrenoceptors as well as imidazoline binding sites. Antagonizing experiments using the highly specific alpha(2)-antagonist methoxy-idazoxan or agmatine (the putative endogenous ligand at imidazoline receptors) were performed to discriminate between putative pharmacological mechanisms involved in the inhibition of cyprid settlement. Agmatine was not able to reverse the effect of any of the tested compounds. However, methoxy-idazoxan almost completely abolished the settlement inhibition mediated by guanabenz (alpha(2)-agonist, I(2) ligand), moxonidine (alpha(2)-agonist, I(1) ligand) and tetrahydrozoline (alpha-agonist, I(2) ligand). The actions of cirazoline (alpha(1)-agonist, I(2) ligand) BU 224 (I(2) ligand) and metrazoline (I(2) ligand) were not reversed by treatment with methoxy-idazoxan. These results suggest that the settlement inhibition evoked by the I(2) ligands and alpha(2)-agonists used in this study of the neurologically simple but well-organized barnacle larva is mediated through different physiological targets important in the overall settlement process.     

Stylochus tauricus,a predator of the barnacle Balanus improvisus in the Black Sea

The flatworm Stylochus tauricus Jacubova has been found associated with the barnacle Balanus improvisus Darwin, on which it feeds. The predation rate (the number of barnacles eaten by one polyclad in a month) ranges between 5–10. Inside the empty shells of B. improvisus some egg-plates of S. tauricus were observed. Pelagic Götte's larvae aged 2–3 days possess 4 lobes while those aged 7–8 days have 5 lobes. Flatworms can prey on the young of another species Balanus eburneus Gould, whereas predation on the mussels Mytilus galloprovincialis Lam. is rare. There is a direct correlation between predator abundance and prey ingested.     

Correlation between surface chemistry and settlement behaviour in barnacle cyprids (Balanus improvisus)

In laboratory-based biofouling assays, the influence of physico-chemical surface characteristics on barnacle settlement has been tested most frequently using the model organism Balanus amphitrite (= Amphibalanus amphitrite). Very few studies have addressed the settlement preferences of other barnacle species, such as Balanus improvisus (= Amphibalanus improvisus). This study aimed to unravel the effects of surface physico-chemical cues, in particular surface-free energy (SFE) and surface charge, on the settlement of cyprids of B. improvisus. The use of well-defined surfaces under controlled conditions further facilitates comparison of the results with recent similar data for B. amphitrite. Zero-day-old cyprids of B. improvisus were exposed to a series of model surfaces, namely self-assembled monolayers (SAMs) of alkanethiols with varying end-groups, homogenously applied to gold-coated polystyrene (PS) Petri dishes. As with B. amphitrite, settlement of cyprids of B. improvisus was influenced by both SFE and charge, with higher settlement on low-energy (hydrophobic) surfaces and negatively charged SAMs. Positively charged SAMs resulted in low settlement, with intermediate settlement on neutral SAMs of similar SFE. In conclusion, it is demonstrated that despite previous suggestions to the contrary, these two species of barnacle show similar preferences in response to SFE; they also respond similarly to charge. These findings have positive implications for the development of novel antifouling (AF) coatings and support the importance of consistency in substratum choice for assays designed to compare surface preferences of fouling organisms.     

Early life-history processes and their implications for the invasion of the barnacle Balanus glandula

As invasions become increasingly prevalent, it is important to understand how spread may be moderated by environmental conditions. This study considered the effect of location and changing substratum temperature on the early life-history processes of the alien barnacle Balanus glandula and its native comparator, Chthamalus dentatus, along the South African coast. Using settlement plates of different colours, temperature was manipulated to assess settlement, mortality, recruitment and growth of the two species. These variables were tracked over 10 weeks using repeat photography. Unexpectedly, there was no evidence of an effect of temperature on early life-history processes of either species. Settlement by the two barnacles was spatially segregated, with B. glandula occurring only on the West Coast where it is the dominant intertidal barnacle, while C. dentatus settled only on the South Coast, which has only recently been invaded by the alien. Despite this, it was notable that the relative settlement of B. glandula on rock was higher than that of C. dentatus. However, the lack of mortality of the native resulted in comparable levels of recruitment among the species. Nonetheless, the propensity of B. glandula to settle sporadically, coupled with fast growth, suggests that the invader may still possess the ability to become dominant along the newly invaded South Coast. This study highlights that measurements of early life-history parameters may not adequately predict the future range and impacts of alien species unless interpreted within the broader context of the nature of the recipient region and species-specific traits of the invader.     

Some effects of early starvation on the survival and development of barnacle nauplii,Balanus improvisus (Darwin)

Newly hatched stage I–II nauplii of Balanus improvisus (Darwin) were “totally starved” (until death) or “partially starved” for the first 48 h and 96 h of their development. Daily mortality and molting were monitored throughout larval development in both starved and fed control groups. Fed control animals exhibited a largely synchronous molting pattern with instars of equal duration. Total starvation suppressed molting beyond stage II; 50% mortality occurred in ≈4 days at both 15 and 21 °C, while longest survival time was 7 days at 15 °C and 6 days at 21 °C. At 15 °C, partially starved nauplii retained the ability to complete naupliar development but at a slower overall rate and with increased mortality relative to controls. These effects were more pronounced in the 96-h group. Increased mortality of stage VI nauplii was evident in both partially starved groups (7.1% for 48 h, 18.8% for 96 h) relative to unstarved controls (3.1%). Stage II nauplii exhibited little resistance to starvation and survival potential may have decreased as soon as 24 h.     

Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin) included in rosin-based coatings

Rosin-based coatings loaded with 0.1% (w/v) ivermectin were found to be effective in preventing colonization by barnacles (Balanus improvisus) both on test panels as well as on yachts for at least two fouling seasons. The leaching rate of ivermectin was determined by mass-spectroscopy (LC/MS-MS) to be 0.7 ng cm^?2 day^?1. This low leaching rate, as deduced from the Higuchi model, is a result of the low loading, low water solubility, high affinity to the matrix and high molar volume of the model biocide. Comparison of ivermectin and control areas of panels immersed in the field showed undisturbed colonisation of barnacles after immersion for 35 days. After 73 days the mean barnacle base plate area on the controls was 13 mm², while on the ivermectin coating it was 3 mm². After 388 days, no barnacles were observed on the ivermectin coating while the barnacles on the control coating had reached a mean of 60 mm². In another series of coated panels, ivermectin was dissolved in a cosolvent mixture of propylene glycol and glycerol formal prior to the addition to the paint base. This method further improved the anti-barnacle performance of the coatings. An increased release rate (3 ng cm^?2 day^?1) and dispersion of ivermectin, determined by fluorescence microscopy, and decreased hardness of the coatings were the consequences of the cosolvent mixture in the paint. The antifouling mechanism of macrocyclic lactones, such as avermectins, needs to be clarified in further studies. Beside chronic intoxication as ivermectin is slowly released from the paint film even contact intoxication occurring inside the coatings, triggered by penetration of the coating by barnacles, is a possible explanation for the mode of action and this is under investigation.     

The complex barnacle perfume: identification of waterborne pheromone homologues in Balanus improvisus and their differential expression during settlement

A key question in barnacle biology is the nature of cues that induce gregarious settlement. One of the characterised cues is the waterborne settlement pheromone (WSP). This study aimed to identify WSP homologues in Balanus improvisus and to investigate their expression during settlement. Six WSP homologues were identified, all containing an N-terminal signal peptide, a conserved core region, and a variable C-terminus comprising several -GR- and -HDDH- motifs. The B. improvisus WSP homologues were expressed in all settlement stages but showed different expression patterns. The homologue most similar to the B. amphitrite WSP was the most abundant and was constantly expressed during settlement. In contrast, several of the other WSP homologues showed the greatest expression in the juvenile stage. The presence of several WSP homologues suggests the existence of a pheromone mix, where con-specificity might be determined by a combination of sequence characteristics and the concentration of the individual components.     

Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin) included in rosin-based coatings

Rosin-based coatings loaded with 0.1% (w/v) ivermectin were found to be effective in preventing colonization by barnacles (Balanus improvisus) both on test panels as well as on yachts for at least two fouling seasons. The leaching rate of ivermectin was determined by mass-spectroscopy (LC/MS-MS) to be 0.7?ng cm(-2) day(-1). This low leaching rate, as deduced from the Higuchi model, is a result of the low loading, low water solubility, high affinity to the matrix and high molar volume of the model biocide. Comparison of ivermectin and control areas of panels immersed in the field showed undisturbed colonisation of barnacles after immersion for 35 days. After 73 days the mean barnacle base plate area on the controls was 13?mm(2), while on the ivermectin coating it was 3?mm(2). After 388 days, no barnacles were observed on the ivermectin coating while the barnacles on the control coating had reached a mean of 60?mm(2). In another series of coated panels, ivermectin was dissolved in a cosolvent mixture of propylene glycol and glycerol formal prior to the addition to the paint base. This method further improved the anti-barnacle performance of the coatings. An increased release rate (3?ng cm(-2) day(-1)) and dispersion of ivermectin, determined by fluorescence microscopy, and decreased hardness of the coatings were the consequences of the cosolvent mixture in the paint. The antifouling mechanism of macrocyclic lactones, such as avermectins, needs to be clarified in further studies. Beside chronic intoxication as ivermectin is slowly released from the paint film even contact intoxication occurring inside the coatings, triggered by penetration of the coating by barnacles, is a possible explanation for the mode of action and this is under investigation.     

The lateral photoreceptor of the barnacle,Balanus eburneus

Summary The lateral eye of the barnacle, Balanus eburneus, fixed in highly concentrated osmium is a lens-shaped body of approximately 250 m in diameter and about 75 m thick. It contains three photoreceptor cells which occupy about 42% of its volume. The photoreceptor cells are irregularly shaped and extend countless dendritic processes which bear rhabdomeres at their ends. Individual rhabdomeres come into contact with rhabdomeres originating from dendrites of the same or of one of the other visual cells. Thirteen per cent of the volume of the photoreceptor cells is taken up by the rhabdomeres. The membranes of the rhabdomeric microvilli contain globular subunits which suggest a 70 Å spacing of rhodopsin molecules. There are two kinds of glial cells. One kind, type A glial cells, makes contact with the fibrous capsule of the photoreceptor. The other kind, type B glial cells, is associated with the photoreceptor cells and extends countless tiny cytoplasmic extensions which interdigitate with similar extensions of the receptor cells. There are approximately 95 type B glial cells and 130 type A glial cells in the receptor. The cytoplasm of the photoreceptor cells contains countless small Golgi fields, mitochondria, microtubules, multivesicular and multilamellar bodies. The extracellular space of the photoreceptor is less than 0.1% of its total volume.The authors wish to thank Mrs. G. Theisen and Miss D. Hupp for expert technical assistance and Drs. M. Behrens, C. Helrich, and C.C. Krischer for many inspiring discussions. This study was partly supported by the SFB 160 of the Deutsche Forschungsgemeinschaft     

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.     

Antifouling activity of the sponge metabolite agelasine D and synthesised analogs on Balanus improvisus

This study reports a screening study for antifouling (AF) activity of the natural compound agelasine D isolated from marine sponges of the genus Agelas and 20 synthesised analogs of agelasines and agelasimines. Agelasine D, together with two of the analogs, ie AV1003A and AKB695, displayed a strong inhibitory effect on settlement of Balanus improvisus cypris larvae. Agelasine D had an EC50 value of 0.11 microM while the two analogs AV1033A and AKB695 had EC50 values of 0.23 and 0.3 microM, respectively. None of these three compounds affected larval mortality as was the case with several of the analogs tested. Moreover, the effect of AV1033A and AKB695 was reversible. When cyprids after 24 h exposure to the compounds were transferred to fresh seawater, the settlement frequency compared with the controls was completely recovered. The properties of the agelasine D analogs AV1003A and AKB695 make them highly attractive candidates as AF agents in future marine coatings.     

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 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.     

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.     

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.     

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.     

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.