Barnacle Cypris Research Articles

Exploring fishery waste potential as antifouling component.

Marine biofouling is a global issue with economic and ecological implications. Existing solutions, such as biocide-based antifouling paints, are toxic for the environment. The search for better antifouling agents remains crucial. Recent research focuses on eco-friendly antifouling paints containing natural compounds like enzymes. This study evaluates enzymatic extracts from fishery residues for antifouling potential. Extracts from Pleoticus muelleri shrimp, Illex argentinus squid, and Lithodes santolla king crab were analyzed. Proteolytic activity and thermal stability were assessed, followed by bioassays on mussel byssus thread formation and barnacle cypris adhesive footprints. All three extracts demonstrated proteolytic activity and 24-h stability at temperate oceanic temperatures, except I. argentinus. P. muelleri extracts hindered cyprid footprint formation and mussel byssus thread generation. Further purification is required for L. santolla extract to assess its antifouling potential activity. This study introduces the use of fishery waste-derived enzyme extracts as a novel antifouling agent, providing a sustainable tool to fight against biofouling formation.

In Vitro Oxidative Crosslinking of Recombinant Barnacle Cyprid Cement Gland Proteins

Barnacle adhesion is a focus for fouling-control technologies as well as the development of bioinspired adhesives, although the mechanisms remain very poorly understood. The barnacle cypris larva is responsible for surface colonisation. Cyprids release cement from paired glands that contain proteins, carbohydrates and lipids, although further compositional details are scant. Several genes coding for cement gland-specific proteins were identified, but only one of these showed database homology. This was a lysyl oxidase-like protein (lcp_LOX). LOX-like enzymes have been previously identified in the proteome of adult barnacle cement secretory tissue. We attempted to produce recombinant LOX in E. coli, in order to identify its role in cyprid cement polymerisation. We also produced two other cement gland proteins (lcp3_36k_3B8 and lcp2_57k_2F5). lcp2_57k_2F5 contained 56 lysine residues and constituted a plausible substrate for LOX. While significant quantities of soluble lcp3_36k_3B8 and lcp2_57k_2F5 were produced in E. coli, production of stably soluble lcp_LOX failed. A commercially sourced human LOX catalysed the crosslinking of lcp2_57k_2F5 into putative dimers and trimers, and this reaction was inhibited by lcp3_36k_3B8. Inhibition of the lcp_LOX:lcp2_57k_2F5 reaction by lcp3_36k_3B8 appeared to be substrate specific, with no inhibitory effect on the oxidation of cadaverine by LOX. The results demonstrate a possible curing mechanism for barnacle cyprid cement and, thus, provide a basis for a more complete understanding of larval adhesion for targeted control of marine biofouling and adhesives for niche applications.

Effect of Multilayer Termination on Nonspecific Protein Adsorption and Antifouling Activity of Alginate-Based Layer-by-Layer Coatings.

Layer-by-layer (LbL) assembly is a versatile platform for applying coatings and studying the properties of promising compounds for antifouling applications. Here, alginate-based LbL coatings were fabricated by alternating the deposition of alginic acid and chitosan or polyethylenimine to form multilayer coatings. Films were prepared with either odd or even bilayer numbers to investigate if the termination of the LbL coatings affects the physicochemical properties, resistance against the nonspecific adsorption (NSA) of proteins, and antifouling efficacy. The hydrophilic films, which were characterized using spectroscopic ellipsometry, water contact angle goniometry, ATR-FTIR spectroscopy, AFM, XPS, and SPR spectroscopy, revealed high swelling in water and strongly reduced the NSA of proteins compared to the hydrophobic reference. While the choice of the polycation was important for the protein resistance of the LbL coatings, the termination mattered less. The attachment of diatoms and settling of barnacle cypris larvae revealed good antifouling properties that were controlled by the termination and the charge density of the LbL films.

Collective behaviours and ambient flow in barnacle cypris larvae

Collective behaviours and ambient flow in barnacle cypris larvae

Shift of Feeding Mode in an Epizoic Stalked Barnacle Inducing Gall Formation of Host Sea Urchin.

SummaryAmong diverse stalked barnacles, Rugilepas pearsei (Thoracica: Cirripedia: Arthropoda) is a rare unique species that is associated with echinoids and has highly atrophied cirri. We rediscovered the barnacle for the first time from description and verified that the barnacles live obligately in half-open galls formed on the test of the sea urchin Echinothrix diadema (Diadematidae: Echinodermata). A molecular phylogenetic analysis demonstrated that the obligate association with echinoids derived from epizoic life on crustaceans. A stable isotope analysis suggests that the barnacle feeds on particulate organic matter (POM) without parasitizing the host echinoids. These findings suggest that the host shift caused losses of plates and feather-like cirri, changes in the attachment device from cementation to anchoring, and a shift in feeding mode from filter feeding to POM collection. The barnacle's epizoic, superficially sub-endozoic, communal life in stout but narrow galls causes repetitive reproduction at the cost of reduced growth.

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.

Surface zooplankton size and taxonomic composition in Bowdoin Fjord, north-western Greenland: A comparison of ZooScan, OPC and microscopic analyses

In Greenland, tidewater glaciers discharge turbid subglacial freshwater into fjords, forming plumes near the calving fronts. To evaluate the effects of this discharge on the zooplankton community in the fjords, we collected sea surface zooplankton samples in Bowdoin Fjord in north-western Greenland during the summer of 2016 and made microscopic, OPC and ZooScan analyses. Within the three quantitative methods, ZooScan has advantages that can evaluate various parameters (e.g., abundance, biomass, size and taxonomic information) simultaneously and has the ability to eliminate abiotic particles, such as silt and sediment, which are abundant in samples. Based on taxonomic biomass data, the zooplankton community is clustered into three groups, which varied spatially: inner, middle and outer fjord groups. Jellyfish dominated the outer fjord group, and barnacle cypris larvae dominated the middle fjord group. For the inner fjord group, large-sized Calanus spp. and chaetognaths were abundant. Since these species are characterized with oceanic taxa, they would intrude through the deep fjord water and subsequently be upwelled through entrainment of glacially modified plume water. From the NBSS analysis on zooplankton size spectra, the steep slope of NBSS in the middle fjord community suggests that the high productivity was caused by the addition of meroplanktonic cypris larvae.

Quantitative analysis of the complete larval settlement process confirms Crisp's model of surface selectivity by barnacles.

For barnacle cypris larvae at the point of settlement, selection of an appropriate surface is critical. Since post-settlement relocation is usually impossible, barnacles have evolved finely tuned surface-sensing capabilities to identify suitable substrata, and a temporary adhesion system for extensive surface exploration. The pattern of exploratory behaviour appears complex and may last for several hours, imposing significant barriers to quantitative measurement. Here, we employ a novel tracking system that enables simultaneous analysis of the larval body movement of multiple individuals over their entire planktonic phase. For the first time, to our knowledge, we describe quantitatively the complete settlement process of cyprids as they explore and select surfaces for attachment. We confirm the 'classic' behaviours of wide searching, close searching and inspection that comprise a model originally proposed by Prof. Dennis Crisp FRS. Moreover, a short-term assay of cyprid body movement has identified inspection behaviour as the best indicator of propensity to settle, with more inspection-related movements occurring in conditions that also promote higher settlement. More than half a century after the model was first proposed by Crisp, there exists a precise method for quantifying cyprid settlement behaviour in wide-ranging investigations of barnacle ecology and applied studies of fouling management.

Diet Composition and Feeding Strategy of the New World SilversideOdontesthes argentinensisin a Temperate Coastal Area (South America)

AbstractThe diet composition and feeding strategy of the New World silversideOdontesthes argentinensiswere evaluated in a temperate coastal area during austral summer. This species is an economically significant resource for the fishing industry in South America. The food preference ofO. argentinensisvaried with its size‐class: the smallest size‐classes preferred diatoms and annelids, larger size‐classes seemed to prefer barnacle cypris larvae, and the largest size‐classes showed a preference for decapod megalopa larvae. Plankton samples were dominated by calanoid and cyclopoid copepods; however, contrary to what was expected,O. argentinensisshowed negative selectivity for both groups. A selectivity index indicated thatO. argentinensisselects the food it eats with regard to prey availability in the environment. The information obtained in this study demonstrates the importance of knowing the food preference ofO. argentinensisto obtain a better condition factor, which will be key to its potential farming in closed systems.

Cirripede Cypris Antennules: How Much Structural Variation Exists Among Balanomorphan Species from Hard-Bottom Habitats?

Barnacle cypris antennules are important for substratum attachment during settlement and on through metamorphosis from the larval stage to sessile adult. Studies on the morphology of cirripede cyprids are mostly qualitative, based on descriptions from images obtained using a scanning electron microscope (SEM). To our knowledge, our study is the first to use scanning electron microscopy to quantify overall structural diversity in cypris antennules by measuring 26 morphological parameters, including the structure of sensory organs. We analyzed cyprids from seven species of balanomorphan barnacles inhabiting rocky shore communities; for comparison, we also included a sponge-inhabiting balanomorphan and a verrucomorphan species. Multivariate analysis of the structural parameters resulted in two distinct clusters of species. From nonmetric multidimensional scaling plots, the sponge-inhabiting Balanus spongicola and Verruca stroemia formed one cluster, while the other balanomorphan species, all from hard bottoms, grouped together in the other cluster. The shape of the attachment disk on segment 3 is the key parameter responsible for the separation into two clusters. The present results show that species from a coastal hard-bottom habitat may share a nearly identical antennular structure that is distinct from barnacles from other habitats, and this finding supports the fact that such species also have rather similar reactions to substratum cues during settlement. Any differences that may be found in settlement biology among such species must therefore be due either to differences in the properties of their adhesive mechanisms or to the way that sensory stimuli are detected by virtually identical setae and processed into settlement behavior by the cyprid.

Instantaneous Flow Structures and Opportunities for Larval Settlement: Barnacle Larvae Swim to Settle.

Water flow affects settlement of marine larvae on several scales. At the smallest scale local flow regime may control the probability of adhesion to the substrate. Our aim was to mechanistically understand the transition from suspended to attached larvae in turbulent flow. Recently it was proposed that opportunities for larval settlement in turbulent boundary layers depend on time windows with suitable instantaneous flow properties. In flume flow we characterized the proportion of suitable time windows in a series of flow velocities with focus on the near-bed flow. The change in the proportion of potential settling windows with increasing free-stream velocities was compared to the proportion of temporary attachment of barnacle cypris larvae at different flow velocities. We found large instantaneous flow variations in the near-bed flow where cyprid attachment took place. The probability of temporary attachment in cyprids declined with local flow speed and this response was compatible with a settling window lasting at least 0.1 s with a maximum local flow speed of 1.9–2.4 cm s-1. Cyprids swam against the near-bed flow (negative rheotaxis) and the swimming speed (1.8 cm s-1) was close to the critical speed that permitted temporary attachment. We conclude that temporary attachment in barnacle cyprids requires upstream swimming to maintain a fixed position relative to the substrate for at least 0.1 s. This behaviour may explain the ability of barnacles to recruit to high-flow environments and give cyprids flexibility in the pre-settlement choice of substrates based on flow regime.

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.

On the morphology of antennular sensory and attachment organs in cypris larvae of the deep‐sea vent/seep barnacles, Ashinkailepas and Neoverruca

Barnacle cypris larvae show high morphological variation in the organs used in search of and attaching to a substratum. This variation may represent adaptation to the habitat of the species. Here, we studied SEM level morphologies of cypris antennular sensory and attachment organs in a deep-sea vent endemic species (Neoverruca sp.) and a vent/seep inhabiting species (Ashinkailepas seepiophila). We compare them with three species from other environments. The antennular morphologies of Neoverruca sp. and A. seepiophila were similar, which is consistent with recent molecular studies showing a close relationship of the two species. The setation pattern of the antennules was very conservative among species from various environments. In contrast, striking differences were observed in the structure of the attachment organ (the third antennular segment). Neoverruca sp. and A. seepiophila had no velum or a skirt surrounding the attachment disc on the third segment, while other cirripede cyprids almost always have either of these structures. In addition, both cyprids of A. seepiophila and Neoverruca sp. had the attachment disc angled toward the substratum, whereas it faces distally in cyprids from hard bottom inhabiting barnacles. We suggest that both velum/skirt and the angle of the attachment disc play an important role, when the antennules are contacting the substratum during surface exploration. Differences in attachment organ structures may be highly adaptive, enabling cirripede species to enter new habitats during evolution.

Resistance of Amphiphilic Polysaccharides against Marine Fouling Organisms.

Amphiphilic coatings are promising candidates for fouling-release applications. As hydrophilic components, polysaccharides are interesting and environmentally benign building blocks. We used covalently coupled alginic acid (AA) and hyaluronic acid (HA) and postmodified them with a hydrophobic fluorinated amine. The surfaces showed good stability under marine conditions and fluorination led to a decreased uptake of Ca(2+) ions after modification. In single species settlement assays (bacteria, diatoms, barnacle cypris larvae), the modification decreased the settlement density and/or the adhesion strength of many of the tested species. Field studies supported findings of the laboratory experiments, as hydrophobic modification of AA and HA decreased diatom colonization.

Barnacle biology before, during and after settlement and metamorphosis: a study of the interface.

Mobile barnacle cypris larvae settle and metamorphose, transitioning to sessile juveniles with morphology and growth similar to that of adults. Because biofilms exist on immersed surfaces on which they attach, barnacles must interact with bacteria during initial attachment and subsequent growth. The objective of this study was to characterize the developing interface of the barnacle and substratum during this key developmental transition to inform potential mechanisms that promote attachment. The interface was characterized using confocal microscopy and fluorescent dyes to identify morphological and chemical changes to the interface and the status of bacteria present as a function of barnacle developmental stage. Staining revealed patchy material containing proteins and nucleic acids, reactive oxygen species amidst developing cuticle, and changes in bacteria viability at the developing interface. We found that as barnacles metamorphose from the cyprid to juvenile stage, proteinaceous materials with the appearance of coagulated liquid were released into and remained at the interface. It stained positive for proteins, including phosphoprotein, as well as nucleic acids. Regions of the developing cuticle and the patchy material itself stained for reactive oxygen species. Bacteria were absent until the cyprid was firmly attached, but populations died as barnacle development progressed. The oxidative environment may contribute to the cytotoxicity observed for bacteria and has the potential for oxidative crosslinking of cuticle and proteinaceous materials at the interface.

Versatile inhibition of marine organism settlement by zwitterionic polymer brushes

The anti-fouling character of polymer brushes against marine organisms was investigated by performing settlement tests with barnacle cypris larvae and mussel larvae in their adhesion period, as well as a marine bacteria colonization test. The surface charge and hydration state in saline seawater appear to be important factors in the versatile anti-fouling performance of zwitterionic polyelectrolyte brushes.

Nontoxic piperamides and their synthetic analogues as novel antifouling reagents

Bioassay-guided isolation of an acetone extract from a terrestrial plant Piper betle produced four known piperamides with potent antifouling (AF) activities, as evidenced by inhibition of settlement of barnacle cypris larvae. The AF activities of the four piperamides and 15 synthesized analogues were compared and their structure–activity relationships were probed. Among the compounds, piperoleine B and 1-[1-oxo-7-(3′,4′-methylenedioxyphenyl)-6E-heptenyl]-piperidine (MPHP) showed strong activity against settlement of cyprids of the barnacle Balanus amphitrite, having EC50 values of 1.1 ± 0.3 and 0.5 ± 0.2 μg ml−1, respectively. No toxicity against zebra fish was observed following incubation with these two compounds. Besides being non-toxic, 91% of piperoleine B-treated cyprids and 84% of MPHP-treated cyprids at a concentration of 100 μM completed normal metamorphosis in recovery bioassays, indicating that the anti-settlement effect of these two compounds was reversible. Hydrolysis and photolysis experiments indicated that MPHP could be decomposed in the marine environment. It is concluded that piperamides are promising compounds for use in marine AF coatings.

Confocal microscopy-based goniometry of barnacle cyprid permanent adhesive

Biological adhesives are materials of particular interest in the fields of bio-inspired technology and antifouling research. The adhesive of adult barnacles has received much attention over the years; however, the permanent adhesive of the cyprid - the colonisation stage of barnacles - is a material about which very little is presently known. We applied confocal laser-scanning microscopy to the measurement of contact angles between the permanent adhesive of barnacle cyprid larvae and self-assembled monolayers of OH- and CH3-terminated thiols. Measurement of contact angles between actual bioadhesives and surfaces has never previously been achieved and the data may provide insight into the physicochemical properties and mechanism of action of these functional materials. The adhesive is a dual-phase system post-secretion, with the behaviour of the components governed separately by the surface chemistry. The findings imply that the cyprid permanent adhesion process is more complex than previously thought, necessitating broad re-evaluation of the system. Improved understanding will have significant implications for the production of barnacle-resistant coatings as well as development of bio-inspired glues for niche applications.

External morphology of barnacle cypris larvae in the family Poecilasmatidae (Cirripedia: Thoracica: Pedunculata): Toward a template for scoring cypris characters

The morphology of the cypris larvae of four genera of pedunculate lepadomorph barnacles of the family Poecilasmatidae (Megalasma, Octolasmis, Poecilasma and Temnaspis) was studied with SEM, focusing on the carapace with lattice organs, thoracopods and abdomen with furcal rami. The morphology of adult poecilasmatid barnacles presents few if any convincing autapomorphies for the family. Cypris characters provide additional data to help confirm the monophyly or paraphyly of the Poecilasmatidae. The form and distribution of the lattice organs and the morphology of the telson with its furcal rami, as well as adult morphology, suggest paraphyly, but we refrain from implementing any cladistic analysis of the Poecilasmatidae based on this limited dataset. Instead, we take the first critical step by formalizing our observations into a preliminary matrix of 10 cypris characters that may be considered as a base for further studies. Several cyprids of Megalasma minus were infested with parasitic benthimermithid nematode larvae.

Effects of poly(l-lactic acid) hydrolysis on attachment of barnacle cypris larvae

Poly(l-lactic acid) (PLLA) applied to immersed solid surfaces in seawater inhibited colonization by barnacles due to the slow-release property of lactic acid. The effect of molecular weight of PLLA on anti-macrofouling activity was confirmed for the first time, with the lowest molecular weight PLLA producing the lowest attaching ratio of cypris larvae of Balanus amphitrite. From the direct addition of lactic acid into a culture of cypris larvae, it was found that the anti-barnacle settlement effect was due to the action of slow-released lactic acid to cypris larvae. The anti-macrofouling function of low molecular weight PLLA was also confirmed in a natural sea environment.