Proteomic comparison of the organic matrices from parietal and base plates of the acorn barnacle Amphibalanus amphitrite.

Acorn barnacles are efficient colonizers on a wide variety of marine surfaces. As they proliferate on critical infrastructure, their settlement and growth have deleterious effects on performance. To address acorn barnacle biofouling, research has focused on the settlement and adhesion processes with the goal of informing the development of novel coatings. This effort has resulted in the discovery and characterization of several proteins found at the adhesive substrate interface, i.e. cement proteins, and a deepened understanding of the function and composition of the biomaterials within this region. While the adhesive properties at the interface are affected by the interaction between the proteins, substrate and mechanics of the calcified base plate, little attention has been given to the interaction between the proteins and the cuticular material present at the substrate interface. Here, the proteome of the organic matrix isolated from the base plate of the acorn barnacle Amphibalanus amphitrite is compared with the chitinous and proteinaceous matrix embedded within A. amphitrite parietal plates. The objective was to gain an understanding of how the basal organic matrix may be specialized for adhesion via an in-depth comparative proteome analysis. In general, the majority of proteins identified in the parietal matrix were also found in the basal organic matrix, including nearly all those grouped in classes of cement proteins, enzymes and pheromones. However, the parietal organic matrix was enriched with cuticle-associated proteins, of which ca 30% of those identified were unique to the parietal region. In contrast, ca 30-40% of the protease inhibitors, enzymes and pheromones identified in the basal organic matrix were unique to this region. Not unexpectedly, nearly 50% of the cement proteins identified in the basal region were significantly distinct from those found in the parietal region. The wider variety of identified proteins in the basal organic matrix indicates a greater diversity of biological function in the vicinity of the substrate interface where several processes related to adhesion, cuticle formation and expansion of the base synchronize to play a key role in organism survival.

Comparative genomics reveals the dynamic evolutionary history of cement protein genes of barnacles from intertidal to deep-sea hydrothermal vents.

Thoracican barnacles are a diverse group of marine organisms for which the availability of genome assemblies is currently limited. In this study, we sequenced the genomes of two neolepadoid species (Ashinkailepas kermadecensis, Imbricaverruca yamaguchii) from hydrothermal vents, in addition to two intertidal species. Genome sizes ranged from 481 to 1054 Mb, with repetitive sequence contents of 21.2% to 50.7%. Concordance rates of orthologs and heterozygosity rates were between 82.4% and 91.7% and between 1.0% and 2.1%, respectively, indicating high genetic diversity and heterozygosity. Based on phylogenomic analyses, we revised the nomenclature of cement genes encoding cement proteins that are not homologous to any known proteins. The major cement gene, CP100A, was found in all thoracican species, including vent-associated neolepadoids, and was hypothesised to be essential for thoracican settlement. Duplicated genes, CP100B and CP100C, were found only in balanids, suggesting potential functional redundancy or acquisition of new functions associated with the calcareous base. An ancestor of CP52 genes was duplicated dynamically among lepadids, pollicipedids with multiple copies on a single scaffold, and balanids with multiple sequential repeats of the conserved regions, but no CP52 genes were found in neolepadoids, providing insights into cement gene evolution among thoracican lineages. This study enhances our understanding of the adhesion mechanisms of thoracicans in underwater environments. The newly sequenced genomes provide opportunities for studying their evolution and ecology, shedding light on their adaptation to diverse marine environments, and contributing to our knowledge of barnacle biology with valuable genomic resources for further studies in this field.

Adaptive Adhesions of Barnacle-Inspired Adhesive Peptides.

The strategy of robust adhesion employed by barnacles renders them fascinating biomimetic candidates for developing novel wet adhesives. Particularly, barnacle cement protein 19k (cp19k) has been speculated to be the key adhesive protein establishing the priming layer in the initial barnacle cement construction. In this work, we systematically studied the sequence design rationale of cp19k by designing adhesive peptides inspired by the low-complexity STGA-rich and the charged segments of cp19k. Combining structure analysis and the adhesion performance test, we found that cp19k-inspired adhesive peptides possess excellent disparate adhesion strategies for both hydrophilic mica and hydrophobic self-assembled monolayer surfaces. Specifically, the low-complexity STGA-rich segment offers great structure flexibility for surface adhesion, while the hydrophobic and charged residues can contribute to the adhesion of the peptides on hydrophobic and charged surfaces. The adaptive adhesion strategy identified in this work broadens our understanding of barnacle adhesion mechanisms and offers valuable insights for designing advanced wet adhesives with exceptional performance on various types of surfaces.

Gene co-option, duplication and divergence of cement proteins underpin the evolution of bioadhesives across barnacle life histories.

Acquisition of new genes often results in the emergence of novel functions and is a key step in lineage-specific adaptation. As a group of sessile crustaceans, barnacles establish permanent attachment through initial cement secretion at the larval phase followed by continuous cement secretion in juveniles and adults. However, the origins and evolution of barnacle larval and adult cement proteins remain poorly understood. By performing microdissection of larval cement glands, transcriptome and shotgun proteomics and immunohistochemistry validation, we identified 30 larval and 27 adult cement proteins of the epibiotic turtle barnacle Chelonibia testudinaria, of which the majority are stage- and barnacle-specific. While only two proteins, SIPC and CP100K, were expressed in both larvae and adults, detection of protease inhibitors and the cross-linking enzyme lysyl oxidase paralogs in larvae and adult cement. Other barnacle-specific cement proteins such as CP100k and CP52k likely share a common origin dating back at least to the divergence of Rhizocephala and Thoracica. Different CP52k paralogues could be detected in larval and adult cement, suggesting stage-specific cement proteins may arise from duplication followed by changes in expression timing of the duplicates. Interestingly, the biochemical properties of larval- and adult-specific CP52k paralogues exhibited remarkable differences. We conclude that barnacle larval and adult cement systems evolved independently, and both emerged from co-option of existing genes and de novo formation, duplication and functional divergence of lineage-specific cement protein genes. Our findings provide important insights into the evolutionary mechanisms of bioadhesives in sessile marine invertebrates.

Biomineralization in Barnacle Base Plate in Association with Adhesive Cement Protein.

Barnacles strongly attach to various underwater substrates by depositing and curing a proteinaceous cement that forms a permanent adhesive layer. The protein MrCP20 present within the calcareous base plate of the acorn barnacle Megabalanus rosa (M. rosa) was investigated for its role in regulating biomineralization and growth of the barnacle base plate, as well as the influence of the mineral on the protein structure and corresponding functional role. Calcium carbonate (CaCO3) growth on gold surfaces modified by 11-mercaptoundecanoic acid (MUA/Au) with or without the protein was followed using quartz crystal microbalance with dissipation monitoring (QCM-D), and the grown crystal polymorph was identified by Raman spectroscopy. It is found that MrCP20 either in solution or on the surface affects the kinetics of nucleation and growth of crystals and stabilizes the metastable vaterite polymorph of CaCO3. A comparative study of mass uptake calculated by applying the Sauerbrey equation to the QCM-D data and quantitative X-ray photoelectron spectroscopy determined that the final surface density of the crystals as well as the crystallization kinetics are influenced by MrCP20. In addition, polarization modulation infrared reflection-absorption spectroscopy of MrCP20 established that, during crystal growth, the content of ß-sheet structures in MrCP20 increases, in line with the formation of amyloid-like fibrils. The results provide insights into the molecular mechanisms by which MrCP20 regulates the biomineralization of the barnacle base plate, while favoring fibril formation, which is advantageous for other functional roles such as adhesion and cohesion.

Mercury-Modulated Immune Responses in Arctic Barnacle Goslings (Branta leucopsis) upon a Viral-Like Immune Challenge.

Historical mining activities in Svalbard (79°N/12°E) have caused local mercury (Hg) contamination. To address the potential immunomodulatory effects of environmental Hg on Arctic organisms, we collected newborn barnacle goslings (Branta leucopsis) and herded them in either a control or mining site, differing in Hg levels. An additional group at the mining site was exposed to extra inorganic Hg(II) via supplementary feed. Hepatic total Hg concentrations differed significantly between the control (0.011 ± 0.002 mg/kg dw), mine (0.043 ± 0.011 mg/kg dw), and supplementary feed (0.713 ± 0.137 mg/kg dw) gosling groups (average ± standard deviation). Upon immune challenge with double-stranded RNA (dsRNA) injection, endpoints for immune responses and oxidative stress were measured after 24 h. Our results indicated that Hg exposure modulated the immune responses in Arctic barnacle goslings upon a viral-like immune challenge. Increased exposure to both environmental as well as supplemental Hg reduced the level of natural antibodies, suggesting impaired humoral immunity. Hg exposure upregulated the expression of proinflammatory genes in the spleen, including inducible nitric oxide synthase (iNOS) and interleukin 18 (IL18), suggesting Hg-induced inflammatory effects. Exposure to Hg also oxidized glutathione (GSH) to glutathione disulfide (GSSG); however, goslings were capable of maintaining the redox balance by de novo synthesis of GSH. These adverse effects on the immune responses indicated that even exposure to low, environmentally relevant levels of Hg might affect immune competence at the individual level and might even increase the susceptibility of the population to infections.

Structure and Composition of the Cuticle of the Goose Barnacle Pollicipes pollicipes: A Flexible Composite Biomaterial.

Arthropods, the largest animal phylum, including insects, spiders and crustaceans, are characterized by their bodies being covered primarily in chitin. Besides being a source of this biopolymer, crustaceans have also attracted attention from biotechnology given their cuticles' remarkable and diverse mechanical properties. The goose barnacle, Pollicipes pollicipes, is a sessile crustacean characterized by their body parts covered with calcified plates and a peduncle attached to a substrate covered with a cuticle. In this work, the composition and structure of these plates and cuticle were characterized. The morphology of the tergum plate revealed a compact homogeneous structure of calcium carbonate, a typical composition among marine invertebrate hard structures. The cuticle consisted of an outer zone covered with scales and an inner homogenous zone, predominantly organic, composed of successive layers parallel to the surface. The scales are similar to the tergum plate and are arranged in parallel and oriented semi-vertically. Structural and biochemical characterization confirmed a bulk composition of ɑ-chitin and suggested the presence of elastin-based proteins and collagen. The mechanical properties of the cuticle showed that the stiffness values are within the range of values described in elastomers and soft crustacean cuticles resulting from molting. The removal of calcified components exposed round holes, detailed the structure of the lamina, and changed the protein properties, increasing the rigidity of the material. This flexible cuticle, predominantly inorganic, can provide bioinspiration for developing biocompatible and mechanically suitable biomaterials for diverse applications, including in tissue engineering approaches.

Chromosome-level genome assembly, annotation, and phylogenomics of the gooseneck barnacle Pollicipes pollicipes.

BACKGROUND: The barnacles are a group of >2,000 species that have fascinated biologists, including Darwin, for centuries. Their lifestyles are extremely diverse, from free-swimming larvae to sessile adults, and even root-like endoparasites. Barnacles also cause hundreds of millions of dollars of losses annually due to biofouling. However, genomic resources for crustaceans, and barnacles in particular, are lacking. RESULTS: Using 62× Pacific Biosciences coverage, 189× Illumina whole-genome sequencing coverage, 203× HiC coverage, and 69× CHi-C coverage, we produced a chromosome-level genome assembly of the gooseneck barnacle Pollicipes pollicipes. The P. pollicipes genome is 770 Mb long and its assembly is one of the most contiguous and complete crustacean genomes available, with a scaffold N50 of 47 Mb and 90.5% of the BUSCO Arthropoda gene set. Using the genome annotation produced here along with transcriptomes of 13 other barnacle species, we completed phylogenomic analyses on a nearly 2 million amino acid alignment. Contrary to previous studies, our phylogenies suggest that the Pollicipedomorpha is monophyletic and sister to the Balanomorpha, which alters our understanding of barnacle larval evolution and suggests homoplasy in a number of naupliar characters. We also compared transcriptomes of P. pollicipes nauplius larvae and adults and found that nearly one-half of the genes in the genome are differentially expressed, highlighting the vastly different transcriptomes of larvae and adult gooseneck barnacles. Annotation of the genes with KEGG and GO terms reveals that these stages exhibit many differences including cuticle binding, chitin binding, microtubule motor activity, and membrane adhesion. CONCLUSION: This study provides high-quality genomic resources for a key group of crustaceans. This is especially valuable given the roles P. pollicipes plays in European fisheries, as a sentinel species for coastal ecosystems, and as a model for studying barnacle adhesion as well as its key position in the barnacle tree of life. A combination of genomic, phylogenetic, and transcriptomic analyses here provides valuable insights into the evolution and development of barnacles.

Effective Synthesis and Antifouling Activity of Dolastatin 16 Derivatives.

Some derivatives of dolastatin 16, a depsipeptide natural product first obtained from the sea hare Dolabella auricularia, were synthesized through second-generation synthesis of two unusual amino acids, dolaphenvaline and dolamethylleuine. The second-generation synthesis enabled derivatizations such as functionalization of the aromatic ring in dolaphenvaline. The derivatives of fragments and whole structures were evaluated for antifouling activity against the cypris larvae of Amphibalanus amphitrite. Small fragments inhibited the settlement of the cypris larvae at potent to moderate concentrations (EC50 = 0.60-4.62 µg/mL), although dolastatin 16 with a substituent on the aromatic ring (24) was much less potent than dolastatin 16.

[The adhesion mechanism of barnacle and its cement proteins: a review].

The adhesive protein secreted by marine sessile animals can resist the resistance of water and exert stickiness under the humid environment. It has become a candidate for the development of high-performance materials in the field of biomedicine and bionics. Barnacles are as one of the marine macrofoulers that can be firmly attached to the underwater substrate materials with different surface characteristics through its cement proteins. To date, the adhesion process of barnacle has been understood in-depth, but the specific underwater adhesion mechanism has not been elucidated and needs further exploration. This review first presented an overview of barnacle and its adhesion process, followed by summarizing the advances of barnacle adhesive protein, its production methods, and applications. Moreover, challenges and future perspectives were prospected.

The adhesion mechanism of barnacle and its cement proteins: a review / 生物工程学报

The adhesive protein secreted by marine sessile animals can resist the resistance of water and exert stickiness under the humid environment. It has become a candidate for the development of high-performance materials in the field of biomedicine and bionics. Barnacles are as one of the marine macrofoulers that can be firmly attached to the underwater substrate materials with different surface characteristics through its cement proteins. To date, the adhesion process of barnacle has been understood in-depth, but the specific underwater adhesion mechanism has not been elucidated and needs further exploration. This review first presented an overview of barnacle and its adhesion process, followed by summarizing the advances of barnacle adhesive protein, its production methods, and applications. Moreover, challenges and future perspectives were prospected.

Comparative analysis of stalked and acorn barnacle adhesive proteomes.

Barnacles interest the scientific community for multiple reasons: their unique evolutionary trajectory, vast diversity and economic impact-as a harvested food source and also as one of the most prolific macroscopic hard biofouling organisms. A common, yet novel, trait among barnacles is adhesion, which has enabled a sessile adult existence and global colonization of the oceans. Barnacle adhesive is primarily composed of proteins, but knowledge of how the adhesive proteome varies across the tree of life is unknown due to a lack of genomic information. Here, we supplement previous mass spectrometry analyses of barnacle adhesive with recently sequenced genomes to compare the adhesive proteomes of Pollicipes pollicipes (Pedunculata) and Amphibalanus amphitrite (Sessilia). Although both species contain the same broad protein categories, we detail differences that exist between these species. The barnacle-unique cement proteins show the greatest difference between species, although these differences are diminished when amino acid composition and glycosylation potential are considered. By performing an in-depth comparison of the adhesive proteomes of these distantly related barnacle species, we show their similarities and provide a roadmap for future studies examining sequence-specific differences to identify the proteins responsible for functional differences across the barnacle tree of life.

Proteogenomic Characterization of the Cement and Adhesive Gland of the Pelagic Gooseneck Barnacle Lepas anatifera.

We focus on the stalked goose barnacle L. anatifera adhesive system, an opportunistic less selective species for the substrate, found attached to a variety of floating objects at seas. Adhesion is an adaptative character in barnacles, ensuring adequate positioning in the habitat for feeding and reproduction. The protein composition of the cement multicomplex and adhesive gland was quantitatively studied using shotgun proteomic analysis. Overall, 11,795 peptide sequences were identified in the gland and 2206 in the cement, clustered in 1689 and 217 proteinGroups, respectively. Cement specific adhesive proteins (CPs), proteases, protease inhibitors, cuticular and structural proteins, chemical cues, and many unannotated proteins were found, among others. In the cement, CPs were the most abundant (80.5%), being the bulk proteins CP100k and -52k the most expressed of all, and CP43k-like the most expressed interfacial protein. Unannotated proteins comprised 4.7% of the cement proteome, ranking several of them among the most highly expressed. Eight of these proteins showed similar physicochemical properties and amino acid composition to known CPs and classified through Principal Components Analysis (PCA) as new CPs. The importance of PCA on the identification of unannotated non-conserved adhesive proteins, whose selective pressure is on their relative amino acid abundance, was demonstrated.

Origin of the biphase nature and surface roughness of biogenic calcite secreted by the giant barnacle Austromegabalanus psittacus.

The calcite grains forming the wall plates of the giant barnacle Austramegabalanus psittacus have a distinctive surface roughness made of variously sized crystalline nanoprotrusions covered by extremely thin amorphous pellicles. This biphase (crystalline-amorphous) structure also penetrates through the crystal's interiors, forming a web-like structure. Nanoprotrusions very frequently elongate following directions related to the crystallographic structure of calcite, in particular, the <- 441> directions, which are the strongest periodic bond chains (PBCs) in calcite. We propose that the formation of elongated nanoprotrusions happens during the crystallization of calcite from a precursor amorphous calcium carbonate (ACC). This is because biomolecules integrated within the ACC are expelled from such PBCs due to the force of crystallization, with the consequent formation of uninterrupted crystalline nanorods. Expelled biomolecules accumulate in adjacent regions, thereby stabilizing small pellicle-like volumes of ACC. With growth, such pellicles become occluded within the crystal. In summary, the surface roughness of the biomineral surface reflects the complex shape of the crystallization front, and the biphase structure provides evidence for crystallization from an amorphous precursor. The surface roughness is generally explained as resulting from the attachment of ACC particles to the crystal surface, which later crystallised in concordance with the crystal lattice. If this was the case, the nanoprotrusions do not reflect the size and shape of any precursor particle. Accordingly, the particle attachment model for biomineral formation should seek new evidence.

Insights into the Synthesis, Secretion and Curing of Barnacle Cyprid Adhesive via Transcriptomic and Proteomic Analyses of the Cement Gland.

Barnacles represent one of the model organisms used for antifouling research, however, knowledge regarding the molecular mechanisms underlying barnacle cyprid cementation is relatively scarce. Here, RNA-seq was used to obtain the transcriptomes of the cement glands where adhesive is generated and the remaining carcasses of Megabalanus volcano cyprids. Comparative transcriptomic analysis identified 9060 differentially expressed genes, with 4383 upregulated in the cement glands. Four cement proteins, named Mvcp113k, Mvcp130k, Mvcp52k and Mvlcp1-122k, were detected in the cement glands. The salivary secretion pathway was significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes, implying that the secretion of cyprid adhesive might be analogous to that of saliva. Lysyl oxidase had a higher expression level in the cement glands and was speculated to function in the curing of cyprid adhesive. Furthermore, the KEGG enrichment analysis of the 352 proteins identified in the cement gland proteome partially confirmed the comparative transcriptomic results. These results present insights into the molecular mechanisms underlying the synthesis, secretion and curing of barnacle cyprid adhesive and provide potential molecular targets for the development of environmentally friendly antifouling compounds.

The Quantitative Proteome of the Cement and Adhesive Gland of the Pedunculate Barnacle, Pollicipes pollicipes.

Adhesive secretion has a fundamental role in barnacles' survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia.

Trace metal biomonitoring in the east Gippsland Lakes estuary using the barnacle Amphibalanus variegatus and mussel Mytilus edulis.

The Gippsland Lakes estuary, a Ramsar listed wetland, in Victoria, Australia, is an area of potential concern for metal pollution due to influxes of human population and associated anthropogenic activities. A biomonitoring exercise was undertaken where the concentrations of 9 metals (Cr, Fe, Cu, Zn, As, Se, Ag, Cd and Hg) were analysed in the soft tissue of two common sessile invertebrates: the mussel Mytilus edulis and the barnacle Amphibalanus variegatus from 6 locations on two different occasions throughout the Gippsland Lakes estuary. A salinity gradient exists in the Lakes, from seawater at Lakes Entrance in the east, decreasing down to < 10 PSU in the west at Lake Wellington during times of rainfall, which is a major factor governing the growth and distribution of both species. Dissolved metal levels in general were low; however, Cu at most sites exceeded the 90% trigger values, while all Zn concentrations exceeded the lowest 80% trigger values of the ANZECC marine water quality guidelines for environmental health. Elevated levels of Cu and Zn were found particularly in barnacles at some sites with environmental contamination due to leaching from anti fouling paints and sacrificial zinc anodes. Elevated levels of Ag and Cd were found in mussels at the Hollands Landing site, which is immediately adjacent to a boat ramp, and Cd and Ag at this site are suspected to originate from inland anthropogenic sources. Concentrations of As in M. edulis across all 6 sites in both sampling periods had mean wet weight As concentrations exceeding the maximum level stated in the FSANZ guidelines. A. variegatus contained elevated levels of Hg especially at the North Arm site with a maximum of 13.6 µg Hg/g dry wt., while A. variegatus also showed temporal changes in Hg concentrations across sites. The maximum Hg concentration found in Mytilus edulis was 1.49 µg Hg/g dry wt. at the Hollands Landing site. Previous contaminant studies of biota in the Lakes have targeted sampling of singular predatory or migratory species, such as Black Bream (Acanthopagrus butcheri) and the Burrunan dolphin (Tursiops australis). This is the first biomonitoring study conducted on sessile organisms to assess metal contamination in the system.

The expression and characterization of recombinant cp19k barnacle cement protein from Pollicipes pollicipes.

Adhesive proteins of barnacle cement have potential as environmentally friendly adhesives owing to their ability to adhere to various substrates in aqueous environments. By understanding the taxonomic breath of barnacles with different lifestyles, we may uncover commonalities in adhesives produced by these specialized organisms. The 19 kDa cement protein (cp19k) of the stalked barnacle Pollicipes pollicipes was expressed in Escherichia coli BL21 to investigate its adhesive properties. Initial expression of hexahistidine-tagged protein (rPpolcp19k-his) yielded low levels of insoluble protein. Co-overproduction of E. coli molecular chaperones GroEL-GroES and trigger factor (TF) increased soluble protein yields, although TF co-purified with the target protein (TF-rPpolcp19k-his). Surface coat analysis revealed high levels of adsorption of the TF-rPpolcp19k-his complex and of purified E. coli TF on both hydrophobic and hydrophilic surfaces, while low levels of adsorption were observed for rPpolcp19k-his. Tag-free rPpolcp19k protein also exhibited low adsorption compared to fibrinogen and Cell-Tak controls on hydrophobic, neutral hydrophilic and charged self-assembled monolayers under surface plasmon resonance assay conditions designed to mimic the barnacle cement gland or seawater. Because rPpolcp19k protein displays low adhesive capability, this protein is suggested to confer the ability to self-assemble into a plaque within the barnacle cement complex. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Sensory receptor repertoire in cyprid antennules of the barnacle Balanus improvisus.

Barnacle settlement involves sensing of a variety of exogenous cues. A pair of antennules is the main sensory organ that the cyprid larva uses to explore the surface. Antennules are equipped with a number of setae that have both chemo- and mechanosensing function. The current study explores the repertoire of sensory receptors in Balanus improvisus cyprid antennules with the goal to better understand sensory systems involved in the settling behavior of this species. We carried out transcriptome sequencing of dissected B. improvisus cyprid antennules. The generated transcriptome assembly was used to search for sensory receptors using HMM models. Among potential chemosensory genes, we identified the ionotropic receptors IR25a, IR8a and IR93a, and several divergent IR candidates to be expressed in the cyprid antennules. We found one gustatory-like receptor but no odorant receptors, chemosensory or odorant-binding proteins. Apart from chemosensory receptors, we also identified 13 potential mechanosensory genes represented by several transient receptor potential channels (TRP) subfamilies. Furthermore, we analyzed changes in expression profiles of IRs and TRPs during the B. improvisus settling process. Several of the sensory genes were differentially expressed during the course of larval settlement. This study gives expanded knowledge about the sensory systems present in barnacles, a taxonomic group for which only limited information about receptors is currently available. It furthermore serves as a starting point for more in depth studies of how sensory signaling affects settling behavior in barnacles with implications for preventing biofouling.

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.