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.

Surveying keratose sponges (Porifera, demospongiae, Dictyoceratida) reveals hidden diversity of host specialist barnacles (Crustacea, Cirripedia, Balanidae).

Sponges represent one of the most species-rich hosts for commensal barnacles yet host utilisation and diversity have not been thoroughly examined. This study investigated the diversity and phylogenetic relationships of sponge-inhabiting barnacles within a single, targeted host group, primarily from Western Australian waters. Specimens of the sponge order Dictyoceratida were surveyed and a total of 64 host morphospecies, representing four families, were identified as barnacle hosts during the study. Utilising molecular (COI, 12S) and morphological methods 42 molecular operational taxonomic units (MOTUs) of barnacles, representing Acasta, Archiacasta, Euacasta and Neoacasta were identified. Comparing inter- and intra-MOTU genetic distances showed a barcode gap between 2.5% and 5% for COI, but between 1% and 1.5% in the 12S dataset, thus demonstrating COI as a more reliable barcoding region. These sponge-inhabiting barnacles were demonstrated to show high levels of host specificity with the majority being found in a single sponge species (74%), a single genus (83%) or a single host family (93%). Phylogenetic relationships among the barnacles were reconstructed using mitochondrial (12S, COI) and nuclear (H3, 28S) markers. None of the barnacle genera were recovered as monophyletic. Euacasta was paraphyletic in relation to the remaining Acastinae genera, which were polyphyletic. Six well-supported clades of molecular operational taxonomic units, herein considered to represent species complexes, were recovered, but relationships between them were not well supported. These complexes showed differing patterns of host usage, though most were phylogenetically conserved with sister lineages typically occupying related hosts within the same genus or family of sponge. The results show that host specialists are predominant, and the dynamics of host usage have played a significant role in the evolutionary history of the Acastinae.

Comparative transcriptomic analysis of deep- and shallow-water barnacle species (Cirripedia, Poecilasmatidae) provides insights into deep-sea adaptation of sessile crustaceans.

BACKGROUND: Barnacles are specialized marine organisms that differ from other crustaceans in possession of a calcareous shell, which is attached to submerged surfaces. Barnacles have a wide distribution, mostly in the intertidal zone and shallow waters, but a few species inhabit the deep-sea floor. It is of interest to investigate how such sessile crustaceans became adapted to extreme deep-sea environments. We sequenced the transcriptomes of a deep-sea barnacle, Glyptelasma gigas collected at a depth of 731 m from the northern area of the Zhongjiannan Basin, and a shallow-water coordinal relative, Octolasmis warwicki. The purpose of this study was to provide genetic resources for investigating adaptation mechanisms of deep-sea barnacles. RESULTS: Totals of 62,470 and 51,585 unigenes were assembled for G. gigas and O. warwicki, respectively, and functional annotation of these unigenes was made using public databases. Comparison of the protein-coding genes between the deep- and shallow-water barnacles, and with those of four other shallow-water crustaceans, revealed 26 gene families that had experienced significant expansion in G. gigas. Functional annotation showed that these expanded genes were predominately related to DNA repair, signal transduction and carbohydrate metabolism. Base substitution analysis on the 11,611 single-copy orthologs between G. gigas and O. warwicki indicated that 25 of them were distinctly positive selected in the deep-sea barnacle, including genes related to transcription, DNA repair, ligand binding, ion channels and energy metabolism, potentially indicating their importance for survival of G. gigas in the deep-sea environment. CONCLUSIONS: The barnacle G. gigas has adopted strategies of expansion of specific gene families and of positive selection of key genes to counteract the negative effects of high hydrostatic pressure, hypoxia, low temperature and food limitation on the deep-sea floor. These expanded gene families and genes under positive selection would tend to enhance the capacities of G. gigas for signal transduction, genetic information processing and energy metabolism, and facilitate networks for perceiving and responding physiologically to the environmental conditions in deep-sea habitats. In short, our results provide genomic evidence relating to deep-sea adaptation of G. gigas, which provide a basis for further biological studies of sessile crustaceans in the deep sea.

Colonization of floats from submerged derelict fishing gears by four protected species of deep-sea corals and barnacles in the Strait of Messina (central Mediterranean Sea).

The distribution of floating litter in marine waters, influenced by currents and wind drag, often determines the dispersal of its encrusting fauna. In the present paper, we observed for the first time the colonization of rafting floats from abandoned, lost or derelict fishing gears (ALDFG) by the four protected deep-sea species: Errina aspera, Desmophyllum pertusum, Madrepora oculata Pachylasma giganteum. Overall, 41 floats, colonized by deep benthic species, were found stranded on the shore of the Sicilian coast of the Strait of Messina, between 2016 and 2019. Species composition, number and occurrence of colonizing organisms were analyzed. On the basis of the species composition (the association between E. aspera, P. giganteum and Megabalanus tulipiformis), the knowledge on their ecology, biogeography, path of local currents, it was possible to define that the area of origin of the most part of these fishing net floats was the Strait of Messina.

Phylogenetic, ecological and biomechanical constraints on larval form: A comparative morphological analysis of barnacle nauplii.

Barnacle naupliar larvae are differentiated from other zooplankton by their unique pair of frontal lateral horns, frontal filaments, and a pear-shaped cephalic shield. Their morphology impose constraints on their ecological functions and reflect their evolutionary history. To explore the potential functional basis underlying the similarities and differences in barnacle larval form, we conducted a meta-analysis on the shape of the barnacle nauplii's cephalic shield and examined its relation to larval size, trophic mode, pelagic larval duration and habitat. Nauplii cephalic shield morphology of 102 species were quantified with normalized elliptic Fourier analysis. Most of the species were distributed around the center of the morphospace but a few extreme groups occupied the periphery: nauplii that were large and lecithotrophic. Subsequent principal component regression analyses showed that larval size was a good predictor of the first shape variations axis (aspect ratio). After allometry adjustment, nauplii from different trophic modes differentiated along the second axis of the major shape variations (relative frontal horn length). Habitat was a poor predictor of variations in naupliar body form, but it could be used to differentiate extreme morphology groups from other nauplii. Our result suggests that size-related biomechanical or developmental constraints and feeding requirements are important in shaping the evolution of the naupliar body form. Within the limitations of these functional constraints, habitat drives the divergence of extreme morphology groups from the majority of species. Our comparative morphometrics analysis demonstrated how variations in larval body form can be quantitatively linked to the functional needs that constrain or drive their diversity, and inform further empirical experiments on larval functional morphology.

Comparative Transcriptomic Analysis Reveals Candidate Genes and Pathways Involved in Larval Settlement of the Barnacle Megabalanus volcano.

Megabalanus barnacle is one of the model organisms for marine biofouling research. However, further elucidation of molecular mechanisms underlying larval settlement has been hindered due to the lack of genomic information thus far. In the present study, cDNA libraries were constructed for cyprids, the key stage for larval settlement, and adults of Megabalanus volcano. After high-throughput sequencing and de novo assembly, 42,620 unigenes were obtained with a N50 value of 1532 bp. These unigenes were annotated by blasting against the NCBI non-redundant (nr), Swiss-Prot, Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Finally, 19,522, 15,691, 14,459, and 10,914 unigenes were identified correspondingly. There were 22,158 differentially expressed genes (DEGs) identified between two stages. Compared with the cyprid stage, 8241 unigenes were down-regulated and 13,917 unigenes were up-regulated at the adult stage. The neuroactive ligand-receptor interaction pathway (ko04080) was significantly enriched by KEGG enrichment analysis of the DEGs, suggesting that it possibly involved in larval settlement. Potential functions of three conserved allatostatin neuropeptide-receptor pairs and two light-sensitive opsin proteins were further characterized, indicating that they might regulate attachment and metamorphosis at cyprid stage. These results provided a deeper insight into the molecular mechanisms underlying larval settlement of barnacles.

Parallel Patterns of Host-Specific Morphology and Genetic Admixture in Sister Lineages of a Commensal Barnacle.

Symbiotic relationships are often species specific, allowing symbionts to adapt to their host environments. Host generalists, on the other hand, have to cope with diverse environments. One coping strategy is phenotypic plasticity, defined by the presence of host-specific phenotypes in the absence of genetic differentiation. Recent work indicates that such host-specific phenotypic plasticity is present in the West Pacific lineage of the commensal barnacle Chelonibia testudinaria (Linnaeus, 1758). We investigated genetic and morphological host-specific structure in the genetically distinct Atlantic sister lineage of C. testudinaria. We collected adult C. testudinaria from loggerhead sea turtles, horseshoe crabs, and blue crabs along the eastern U.S. coast between Delaware and Florida and in the Gulf of Mexico off Mississippi. We find that shell morphology, especially shell thickness, is host specific and comparable in similar host species between the Atlantic and West Pacific lineages. We did not detect significant genetic differentiation related to host species when analyzing data from 11 nuclear microsatellite loci and mitochondrial sequence data, which is comparable to findings for the Pacific lineage. The most parsimonious explanation for these parallel patterns between distinct lineages of C. testudinaria is that C. testudinaria maintained phenotypic plasticity since the lineages diverged 4-5 mya.

Automated tracking and classification of the settlement behaviour of barnacle cyprids.

A focus on the development of nontoxic coatings to control marine biofouling has led to increasing interest in the settlement behaviour of fouling organisms. Barnacles pose a significant fouling challenge and accordingly the behaviour of their settlement-stage cypris larva (cyprid) has attracted much attention, yet remains poorly understood. Tracking technologies have been developed that quantify cyprid movement, but none have successfully automated data acquisition over the prolonged periods necessary to capture and identify the full repertoire of behaviours, from alighting on a surface to permanent attachment. Here we outline a new tracking system and a novel classification system for identifying and quantifying the exploratory behaviour of cyprids. The combined system enables, for the first time, tracking of multiple larvae, simultaneously, over long periods (hours), followed by automatic classification of typical cyprid behaviours into swimming, wide search, close search and inspection events. The system has been evaluated by comparing settlement behaviour in the light and dark (infrared illumination) and tracking one of a group of 25 cyprids from the water column to settlement over the course of 5 h. Having removed a significant technical barrier to progress in the field, it is anticipated that the system will accelerate our understanding of the process of surface selection and settlement by barnacles.

Darwin's "Mr. Arthrobalanus": Sexual Differentiation, Evolutionary Destiny and the Expert Eye of the Beholder.

Darwin's Cirripedia project was an exacting exercise in systematics, as well as an encrypted study of evolution in action. Darwin had a long-standing interest and expertise in marine invertebrates and their sexual arrangements. The surprising and revealing sexual differentiation he would uncover amongst barnacles represented an important step in his understanding of the origins of sexual reproduction. But it would prove difficult to reconcile these findings with his later theorizing. Moreover, the road to discovery was hardly straightforward. Darwin was both helped and hindered by the tacit expectations generated by his transformist theorizing, and had to overcome culturally-embedded assumptions about gender and reproductive roles. Significant observational backtracking was required to correct several oversights and misapprehensions, none more so than those relating to the chronically misunderstood "Mr. Arthrobalanus." With careful attention to chronology, this paper highlights some curious and overlooked aspects of Darwin's epic project.

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.

Ecology and biogeography of megafauna and macrofauna at the first known deep-sea hydrothermal vents on the ultraslow-spreading Southwest Indian Ridge.

The Southwest Indian Ridge is the longest section of very slow to ultraslow-spreading seafloor in the global mid-ocean ridge system, but the biogeography and ecology of its hydrothermal vent fauna are previously unknown. We collected 21 macro- and megafaunal taxa during the first Remotely Operated Vehicle dives to the Longqi vent field at 37° 47'S 49° 39'E, depth 2800 m. Six species are not yet known from other vents, while six other species are known from the Central Indian Ridge, and morphological and molecular analyses show that two further polychaete species are shared with vents beyond the Indian Ocean. Multivariate analysis of vent fauna across three oceans places Longqi in an Indian Ocean province of vent biogeography. Faunal zonation with increasing distance from vents is dominated by the gastropods Chrysomallon squamiferum and Gigantopelta aegis, mussel Bathymodiolus marisindicus, and Neolepas sp. stalked barnacle. Other taxa occur at lower abundance, in some cases contrasting with abundances at other vent fields, and δ13C and δ15N isotope values of species analysed from Longqi are similar to those of shared or related species elsewhere. This study provides baseline ecological observations prior to mineral exploration activities licensed at Longqi by the United Nations.

Morphology of Cyprid Attachment Organs Compared Across Disparate Barnacle Taxa: Does It Relate to Habitat?

This study used morphometric analyses to compare the structure of the third antennular segment, also called the attachment organ, in cyprid larvae from cirripede species representing a diverse set of taxonomic groups. The aim was to investigate the degree of morphological variation in view of the diversity of habitats, settlement substrata, and modes of life found in the Cirripedia. In all cyprids the third segment features a flat surface (the attachment disc) covered with small cuticular villi thought to function in adhesion. The parameters analyzed were the angle of this disc relative to the long axis of the antennule, its shape (outline), the density of cuticular villi, and the type of cuticular structure encircling the disc. The 10 species studied came from most major groups of cirripedes, and comprised shallow-water forms inhabiting hard bottoms (Capitulum mitella, Pollicipes pollicipes, Semibalanus balanoides, Austrominius modestus, Megabalanus rosa), sublittoral forms (Verruca stroemia, Scalpellum scalpellum), epibiotic forms settling on live, soft tissues (Balanus spongicola, Savignium crenatum), and a parasite (Peltogaster paguri). Significant structural variation was found among the species, but due to limited taxon sampling it was unclear whether the differences relate to ecological factors or phylogenetic affiliation. The disc perimeter is guarded by either a series of long and thin cuticular fringes overreaching the rim of the disc (= a velum) or a few low, but very broad cuticular flaps (= a skirt). The presence of a velum (in all rocky-shore species) or a skirt (all other species) around the attachment disc was the only parameter that was clearly correlated with habitat. The shape of the third antennular segment varied from a symmetrical bell shape with a distally facing attachment disc having a circular disc outline, to segments that were elongated in side view, with a very tilted ventral disc surface having an elliptical disc outline. The bell shape may be most common in forms from rocky shores, but in our test of morphometric parameters only Scalpellum scalpellum (sublittoral), Savignium crenatum (epibiotic in corals), and Peltogaster paguri (parasitic) had shapes that differed significantly from the other species. The density of villi on the attachment disc varied significantly, but also showed no clear-cut correlation with substratum or habitat. Attachment organ structure is clearly the most variable feature in cirripede cyprids. To evaluate the degree to which attachment organ structure is correlated with habitat, settlement substratum, and mode of life, future studies should employ a more refined statistical analysis on an enlarged dataset, with much increased taxon sampling and a more multifaceted definition of ecological variables.

Revision of the coral-inhabiting genus Conopea (Cirripedia: Archaeobalanidae) with description of two new species of the genera Conopea and Acasta.

The morphology of archaeobalanid barnacles of the genera Conopea and Acasta inhabiting cnidarians of the orders Alcyonacea and Antipatharia was surveyed. Based on morphological characteristics, it became evident that the species of the nominal genus Conopea fell into three natural groups affiliated to three archaeobalanid genera, Conopea s.s., Acasta and Solidobalanus. The relationships between the species of Conopea s.l. and those of Acasta inhabiting alcyanaceans are analyzed using a cladistic approach. The barnacles of the genus Conopea s.s. are characterized by a strong, firm shell; the orifice is not dentate; rostral and sometimes carinal plates are often elongated in their basal parts; the rostro-carinal axis of the basis is often elongated and clasps the axis of the host coral; the radii have summits parallel to the basal margin of the parietes, and denticulated sutural margins; the scutum has simple growth ridges without longitudinal striation or ribs; the basitergal angle is truncated (sinusoid); and the basidorsal point of the penis is developed. The genus Conopea s.s. encompasses 20 epizoic species from tropical and temperate seas, inhabiting alcyonaceans (sea fans or gorgonians) and antipatharians. A new species of Conopea and a new species of Acasta are described, and a key to the species of Conopea s.s. is provided.

Acasta sulcata akanthosa, new replacement name for Acasta sulcata spinosa.

Acasta sulcata spinosa Daniel, 1955 has been discovered to be a junior homonym of Acasta spinosa Hiro, 1939. As both names were originally described in the genus Acasta, ICZN (International Commission on Zoological Nomenclature) Article 57.2 defines A. sulcata spinosa as a primary junior homonym, even though it is of subspecific nature.

On the authorship of Austromegabalanus psittacus (Molina).

Austromegabalanus psittacus is a large, common and economically important shoreline barnacle species of the west coast of South America. It was originally described by Juan Ignacio Molina as Lepas psittacus, but some confusion has arisen as to the exact reference to and year of publication of this species.

The whale barnacle Cryptolepas rhachianecti (Cirripedia: Coronulidae), a phoront of the grey whale Eschrichtius robustus (Cetacea: Eschrichtiidae), from a sandy beach in The Netherlands.

An isolated compartment of a whale barnacle is herein described from Recent beach deposits in Zoutelande (Walcheren, The Netherlands). This specimen is identified as belonging to the extant coronulid species Cryptolepas rhachianecti, currently known as an epizoic symbiont of the grey whale Eschrichtius robustus. This find represents the first occurrence of C. rhachianecti outside the North Pacific, and the first one as a (sub)fossil. In view of the fact that E. robustus, which is currently confined to the North Pacific, is known as a subfossil from the northeastern Atlantic between late Late Pleistocene (c. 45,000 years ago) and historical (c. 1700 AD) times, we propose a similar (late Quaternary) age for the isolated compartment. The find indicates that the extinct late Quaternary northeastern Atlantic population of E. robustus was infected by Cryptolepas rhachianecti. Our find is, therefore, compatible with the hypothesis of an ancient grey whale migration route running between the subtropical/temperate waters of the northeast Atlantic (or Mediterranean Basin), and the cold waters of the Baltic Sea (or southern Arctic Ocean), through the southern North Sea. Finally, we discuss the systematic placement of the fossil barnacle species Cryptolepas murata and propose the possibility of its removal from the genus Cryptolepas pending further investigations.

Ecology and Evolution of Phenotypic Plasticity in the Penis and Cirri of Barnacles.

Most barnacles are sessile, simultaneous hermaphrodites that reproduce by copulation. This is achieved through the extension of a muscular penis, famous for being the proportionally largest in the animal kingdom. The penis is a long cylindrical or conical organ, composed of a series of folded rings, allowing it to stretch to great lengths. The penises are covered with chemosensory setae allowing them to seek out receptive neighbors. For many species, the condition of the penis changes seasonally. In the most extreme circumstances, it degenerates and is shed during the first post-mating molt and is re-grown for the next mating season. Barnacle penises have been shown to exhibit phenotypic plasticity in response to many different challenges. When exposed to heavy waves, diameter is increased by thickening both the cuticle and muscles. When mates are far, length increases by adding ringed annulations. Experiments have shown that these plastic traits are modular, capable of changing independently from each other and that they improve mating ability. Alternate strategies to increase reproductive ability by barnacles include the production of dwarf and complemental males, sperm casting and sperm leakage, and aerial copulation. All of these mating strategies may have important implications for the study of reproductive biology, life history, and sex allocation theory.

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.