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.     

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.     

Investigating a possible role for the bacterial signal molecules N‐acylhomoserine lactones in Balanus improvisus cyprid settlement

Increased settlement on bacterial biofilms has been demonstrated for a number of marine invertebrate larvae, but the nature of the cue(s) responsible is not well understood. We tested the hypothesis that the bay barnacle Balanus improvisus utilizes the bacterial signal molecules N‐acylhomoserine lactones (AHLs) as a cue for the selection of sites for permanent attachment. Single species biofilms of the AHL‐producing bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. BR1 were attractive to settling cypris larvae of B. improvisus. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL‐degrading gene (aiiA), the ability of the bacteria to attract cyprids was abolished. In addition, cyprids actively explored biofilms of E. coli expressing the recombinant AHL synthase genes luxI from Vibrio fischeri (3‐oxo‐C6‐HSL), rhlI from Pseudomonas aeruginosa (C4‐HSL/C6‐HSL), vanI from V. anguillarum (3‐oxo‐C10‐HSL) and sulI from Sulfitobacter sp. BR1 (C4‐HSL, 3‐hydroxy‐C6‐HSL, C8‐HSL and 3‐hydroxy‐C10‐HSL), but not E. coli that did not produce AHLs. Finally, synthetic AHLs (C8‐HSL, 3‐oxo‐C10‐HSL and C12‐HSL) at concentrations similar to those found within natural biofilms (5 μm ) resulted in increased cyprid settlement. Thus, B. improvisus cypris exploration of and settlement on biofilms appears to be mediated by AHL‐signalling bacteria in the laboratory. This adds to our understanding of how quorum sensing inhibition may be used as for biofouling control. Nonetheless, the significance of our results for larvae settling naturally in the field, and the mechanisms that underlay the observed responses to AHLs, is as yet unknown.     

Possible molecular mechanisms of species recognition by barnacle larvae inferred from multi-specific sequencing analysis of proteinaceous settlement-inducing pheromone

Gregarious settlement is essential for reproduction and survival of many barnacles. A glycoprotein, settlement-inducing protein complex (SIPC) has been recognized as a signal for settlement and it is expressed in both conspecific adults and larvae. Although the settlement-inducing activities of SIPC are species-specific, the molecular-based mechanism by which larvae distinguish conspecific SIPC from the SIPC of other species is still unknown. Here, the complete primary structure of the SIPC of Megabalanus coccopoma, as well as the partial structure of the SIPCs of Balanus improvisus, Megabalanus rosa, and Elminius modestus are reported. These SIPCs contain highly variable regions that possibly modulate the affinity for the receptor, resulting in the species specificity of SIPC. In addition, the distribution patterns of potential N-glycosylation sites were seen to be different among the various species. Differences in such post-translational modifications may contribute to the species specificity of SIPC.     

Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid‐phase IEF sample prefractionation combined with 2‐DE and MALDI‐TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium‐binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required.     

Adding coral rubble to substrata enhances settlement of Pocillopora?damicornis larvae

Settlement preferences of Pocillopora damicornis larvae were examined on artificial substrata. Planulation of P. damicornis followed a lunar cycle and the release of larvae occurred after new moon. P. damicornis larvae had the highest rates of settlement within 3 days of being presented settlement substrata. Cumulative settlement gradually increased from 3 to 8 days, and post-settlement mortality was most frequent after 8 days. Settlement experiments showed greatest settlement preference to cement tiles containing 10% coral rubble. This study suggests that physical cues are important in the settlement process, which may be useful for coral reef rehabilitation projects.     

Serum antibody responses to Wolbachia surface protein in patients with human lymphatic filariasis

Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B‐cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T‐cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR‐amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T‐7 expression vector pRSET‐A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP‐specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP‐specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of B‐ and T‐cell epitopes. Hence, a combinatorial approach of in silico predictions and wet‐lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.     

Chemical settlement inhibition versus post-settlement mortality as an explanation for differential fouling of two congeneric seaweeds

It has been proposed that seaweed secondary metabolites, e.g. brown algal phlorotannins, may have an ecologically important function as a chemical defence against epiphytes, by acting against colonisation of epiphytic organisms. We tested whether the low epiphytic abundance on the invasive brown seaweed Fucus evanescens, compared to the congeneric F. vesiculosus, is due to a more effective chemical defence against epiphyte colonisation. A field survey of the distribution of the common fouling organism Balanus improvisus (Cirripedia) showed that the abundance was consistently lower on F. evanescens than on F. vesiculosus. However, contrary to expectations, results from experimental studies indicated that F. vesiculosus has a more effective anti-settlement defence than F. evanescens. In settlement experiments with intact fronds of the two Fucus species, both species deterred settlement by barnacle larvae, but settlement was lower on F. vesiculosus both in choice and no-choice experiments. Phlorotannins from F. vesiculosus also had a stronger negative effect on larval settlement and were active at a lower concentration than those from F. evanescens. The results show that Fucus phlorotannins have the potential to inhibit settlement of invertebrate larvae, but that settlement inhibition cannot explain the lower abundance of the barnacle Balanus improvisus on F. evanescens compared to F. vesiculosus. Assessment of barnacle survival in the laboratory and in the field showed that this pattern could instead be attributed to a higher mortality of newly settled barnacles. Observation suggests that the increased mortality was due to detachment of young barnacles from the seaweed surface. This shows that the antifouling mechanism of F. evanescens acts on post-settlement stages of B. improvisus.     

Correlative assays of barnacle cyprid behaviour for the laboratory evaluation of antifouling coatings: a study of surface energy components

Laboratory evaluation of antifouling coatings is underpinned by settlement studies with specific fouling organisms. Established methods provide insight into the likelihood of failure of a particular coating system, but can neglect the process of surface selection that often precedes attachment. The present approach for quantifying the exploratory behaviour of barnacle cypris larvae suggested that inspection behaviour can be a rapid and predictive proxy for settlement. Two series of xerogels with comparable total surface energy, but different dispersive and polar components, were evaluated. Settlement assays with three-day-old cyprids of Balanus improvisus demonstrated that while attachment was not linked directly to dispersive free energy, the composition of the xerogel was nevertheless significant. Behavioural analysis provided insight into the mechanism of surface rejection. In the case of a 50:50?PH/TEOS (phenyltriethoxysilane-based) xerogel vs a 50:50 TFP/TEOS (3,3,3-trifluoropropyltrimethoxysilane-based) xerogel, wide-searching behaviour was absent on the former.     

Biofilm history and oxygen availability interact to affect habitat selection in a marine invertebrate

In marine systems, oxygen availability varies at small temporal and spatial scales, such that current oxygen levels may not reflect conditions of the past. Different studies have shown that marine invertebrate larvae can select settlement sites based on local oxygen levels and oxygenation history of the biofilm, but no study has examined the interaction of both. The influence of normoxic and hypoxic water and oxygenation history of biofilms on pre-settlement behavior and settlement of the bryozoan Bugula neritina was tested. Larvae used cues in a hierarchical way: the oxygen levels in the water prime larvae to respond, the response to different biofilms is contingent on oxygen levels in the water. When oxygen levels varied throughout biofilm formation, larvae responded differently depending on the history of the biofilm. It appears that B. neritina larvae integrate cues about current and historical oxygen levels to select the appropriate microhabitat and maximize their fitness.     

Development in an estuarine fouling community: The influence of early colonists on later arrivals

Summary Experiments were performed to determine if earlier colonists inhibited, enhanced, or were necessary for establishment of later colonists during development of an estuarine fouling community at Lewes, Delaware. We determined the significance of earlier stages on the successional process by functionally removing early colonizing species. Since settlement of sessile invertebrates onto our experimental test plates was seasonal, we were able to accomplish functional removal of early colonists by putting out clean test panels after these species had ceased settling. Comparisons between panels initially submerged at three different times in 1974 and 1975, and between panels put out at one-month intervals throughout the study (to describe seasonal settlement patterns) allowed us to determine interactions between adult populations of earlier colonists and colonizing individuals of later arriving species.The dominant sessile species in our system and their times of settlement were: a barnacle (Balanus improvisus) — April through June, a polychaete (Hydroides dianthus) — July and August, a tunicate (Molgula manhatensis) — June through October, a hydroid (Tubularia crocea) — July through October, and a mussel (Mytilus edulis) — November through April. All successional series eventually came to be dominated by M. edulis, and it persisted as the dominant for over a year.A variety of species interactions were observed. M. edulis inhibited colonization by all other dominants and B. improvisus partially inhibited settlement of M. manhattensis. The presence of adult M. manhattensis had no influence on summer settlement of T. crocea, but the hydroids enhanced settlement of tunicates in the fall. During both years of our study, larger settlements of mussels were noted on panels harboring tunicates and hydroids than on bare surfaces. H. dianthus, on the other hand, became established only on bare substrates, and colonization was almost totally inhibited by other dominants.Development in our fouling community did not conform to any single model of community development presented to date. Instead, components of several models were observed within our relatively simple (in terms of number of species) system. For example, facilitation (enhancement of later colonists by earlier ones) and inhibition (resistance of earlier colonists to invasion by later colonists) were both observed. However, we found no evidence earlier colonists were essential for establishment of the next developmental stage. In fact, inhibitory interactions appeared to be much more prevalent than facilitative interactions. The former may also have more profound effects on community development since they more often determine eventual species compositions.     

Predation risk assessment by larval reef fishes during settlement-site selection

Predation rates of marine species are often highest during the transition from the pelagic to the benthic life stage. Consequently, the ability to assess predation risk when selecting a settlement site can be critical to survival. In this study, pairwise choice trials were used to determine whether larvae of three species of anemonefish (Amphiprion melanopus, A. percula and Premnas biaculeatus) are able to (1) assess the predation risk of potential anemone settlement sites through olfactory cues alone and (2) alter their settlement choices depending on the options available (host or non-host anemone). When predation risk was assessed with host and non-host anemone species independently, all species of anemonefish significantly chose the odor associated with the low-risk settlement option over the high-risk site. Most importantly, all species of anemonefish selected water with olfactory cues from their host anemone regardless of predation risk when paired against non-host anemone odor. These results demonstrate that larval reef fishes can use olfactory cues for complex risk assessment during settlement-site selection; however, locating the correct habitat is the most important factor when selecting a settlement site.     

Adrenoceptor Compounds Prevent the Settlement of Marine Invertebrate Larvae: Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) and Hydroides elegans (Polychaeta)

The effects of the neurotransmitter blockers idazoxan and phentolamine on the larval settlement of three marine invertebrate species belonging to three different phyla were investigated by using in vitro concentration-response bioassays. Since neurotransmitters are known to influence metamorphic transitions in invertebrate larvae, neurotransmitter blockers were tested to evaluate their sublethal effects on larvae. The α-adrenergic antagonists idazoxan and phentolamine inhibited settlement of Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) larvae, and larvae of the polychaete Hydroides elegans (Polychaeta) in a concentration- and taxon-dependent manner. At concentrations of 10^?3 M of both agents, larvae of all three species became immobile and subsequently died within 24 h. While cumulative settlement rates were observed after 48 h for B. amphitrite and H. elegans, and after 5 h for B. neritina, > 90% of the larvae that settled did so within 24 h for the first two species and within 1 h for B. neritina. The tendency of the hydrophobic idazoxan and phentolamine to accumulate at solid surfaces most probably contributes to their successful inhibition of larval settlement. This ability makes them particularly attractive as candidates for the development of slow-release carriers in antifouling paints.     

New pollen-specific receptor kinases identified in tomato,maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes

We previously characterized LePRK1 and LePRK2, pollen-specific receptor kinases from tomato (Muschietti et al., 1998). Here we identify a similar receptor kinase from maize, ZmPRK1, that is also specifically expressed late in pollen development, and a third pollen receptor kinase from tomato, LePRK3. LePRK3 is less similar to LePRK1 and LePRK2 than either is to each other. We used immunolocalization to show that all three LePRKs localize to the pollen tube wall, in partially overlapping but distinct patterns. We used RT-PCR and degenerate primers to clone homologues of the tomato kinases from other Solanaceae. We deduced features diagnostic of pollen receptor kinases and used these criteria to identify family members in the Arabidopsis database. RT-PCR confirmed pollen expression for five of these Arabidopsis candidates; two of these are clearly homologues of LePRK3. Our results reveal the existence of a distinct pollen-specific receptor kinase gene family whose members are likely to be involved in perceiving extracellular cues during pollen tube growth.     

Charged hydrophilic polymer brushes and their relevance for understanding marine biofouling

The resistance of charged polymers to biofouling was investigated by subjecting cationic (PDMAEMA), anionic (PSPMA), neutral (PHEMA-co-PEG₁₀MA), and zwitterionic (PSBMA) brushes to assays testing protein adsorption; attachment of the marine bacterium Cobetia marina; settlement and adhesion strength of zoospores of the green alga Ulva linza; settlement of barnacle (Balanus amphitrite and B. improvisus) cypris larvae; and field immersion tests. Several results go beyond the expected dependence on direct electrostatic attraction; PSPMA showed good resistance towards attachment of C. marina, low settlement and adhesion of U. linza zoospores, and significantly lower biofouling than on PHEMA-co-PEG₁₀MA or PSBMA after a field test for one week. PDMAEMA showed potential as a contact-active anti-algal coating due to its capacity to damage attached spores. However, after field testing for eight weeks, there were no significant differences in biofouling coverage among the surfaces. While charged polymers are unsuitable as antifouling coatings in the natural environment, they provide valuable insights into fouling processes, and are relevant for studies due to charging of nominally neutral surfaces.     

HABITAT DIFFERENCES IN THE TIMING OF REPRODUCTION OF THE INVASIVE ALGA SARGASSUM MUTICUM (PHAEOPHYTA,SARGASSACEAE) OVER TIDAL AND LUNAR CYCLES1

Sargassum muticum (Yendo) Fensholt is an invasive species that is firmly established on intertidal and subtidal rocky shores of Europe and the Pacific coast of North America. Local success and spread of S. muticum is thought to rely on its reproductive potential that seems dependent on exogenous factors like tidal and lunar cycles. This study is the first to compare the reproductive patterns (periodicity of egg expulsion and embryo settlement) of this invader in two different habitats: the middle and low intertidal. The combination of monthly, daily, and tidal samples at triplicate sites within each habitat showed a semilunar periodicity of egg expulsion and embryo settlement coincident with increasing tidal amplitude just before full and new moons. In both habitats, duration of each egg expulsion event was ～1 week, and embryo settlement occurred during the first daily low tide and with the incoming high tide during spring tides. However, both expulsion and settlement started 1–2 d earlier, expulsion saturation was faster, and settlement was higher in the mid‐ compared to the low intertidal. Our results suggest that the exact timing of gamete expulsion and embryo release of S. muticum responds to local factors, including tidal cues, which result in differences between mid‐ and low‐intertidal habitats.     

Do settlement dynamics influence competitive interactions between an alien tunicate and its native congener?

Variation in density of early stages, that is, larvae and juveniles, is a major determinant of the distribution and abundance of the adult population of most marine invertebrates. These early stages thus play a key role in competitive interactions, and, more specifically, in invasion dynamics when biologically similar native and non‐native species (NNS) come into contact in the same habitat. We examined the settlement dynamics and settlement rate of two important members of the fouling community that are common on human‐made infrastructures around the world: Ciona robusta (formerly known as Ciona intestinalis type A) and C. intestinalis (formerly known as C. intestinalis type B). In the western English Channel, the two species live in close syntopy following the recent introduction of C. robusta in the native European range of C. intestinalis. Using settlement panels replaced monthly over 2 years in four marinas (including one studied over 4 years) and species‐diagnostic molecular markers to distinguish between juveniles of both species (N = 1,650), we documented similar settlement dynamics of both species, with two settlement periods within a calendar year. With one exception, settlement times were highly similar in the congeners. Although the NNS showed lower settlement density than that of the native congener, its juvenile recruitment was high during the second settlement period that occurs after the warm season, a pattern also observed in adult populations. Altogether, our results suggest that species’ settlement dynamics do not lead to the dominance of one species over the other through space monopolization. In addition, we showed that changes over time are more pronounced in the NNS than in the native species. This is possibly due to a higher sensitivity of the NNS to changes of environmental factors such as temperature and salinity. Environmental changes may thus eventually modify the strength of competitive interactions between the two species as well as species dominance.     

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.