Specialised rootlets of Sacculina pilosella (Rhizocephala: Sacculinidae) used for interactions with its host's nervous system.

Parasitic rhizocephalan barnacles induce morphological, physiological, and behavioural changes in their hosts. The mechanisms of these intimate host-parasite interactions remain unknown. We have shown previously that rootlets of the internae of Peltogasterella gracilis and Peltogaster paguri penetrate the ganglion's envelope of their hermit crab hosts and form specialised structures in the ganglion periphery, the so-called goblet-shaped organs. Here, we examine the gross morphology and ultrastructure of these goblet-shaped organs in the interna of Sacculina pilosella. They consist of three layers of cells; in the intermediate layer of the organs, unusual lamellar bodies and muscle cells were found. Extensive degeneration of the host nervous tissue was observed in the funnel of the goblet-shaped organs. We conclude that the ability to penetrate into the host's nervous tissue could be a common trait in rhizocephalans. The goblet-shaped organs may play a key role in the host-parasite relationships by enabling the parasite to influence the host via hormones and neurotransmitters.

Permanently Fused Setules Create Unusual Folding Fans Used for Swimming in Cyprid Larvae of Barnacles.

Many crustacean swimming appendages carry arrays of plumose setae-exoskeletal, feather-like structures of long bristles (setae) with short branches (setules) distributed along two sides. Although closely spaced, setae are not physically interconnected. Setal arrays function during swimming as drag-based leaky paddles that push the organism through water. Barnacle cyprids, the final, non-feeding larval stage, swim with six pairs of legs (thoracopods) that open and close setal arrays in alternating high-drag power strokes and low-drag recovery strokes. While studying cyprid swimming, we found that their thoracopods contained setae permanently cross-linked by fused setules. These cuticular connections would seem highly unlikely because setae are individually produced exoskeletal secretions, and the connections imply unknown processes for the production or modification of crustacean setae. We describe the morphology and function of plumose setae on cyprids of Balanus glandula and other species across the clade Cirripedia. Setules from adjacent plumose setae are seamlessly joined at their tips and occur in three distinct linkage patterns. Thoracopods lack muscles to open and close the array; interconnected setae are instead pulled apart, producing a paddle-like fan with high drag when appendages spread laterally during power strokes. Setules are spring-like, passively closing setae into tight bundles with low drag during recovery strokes. The linked setules occur in the three main clades of the Cirripedia. This cuticular arrangement is effective in swimming, may eliminate the need for muscles to close the setal array, and may represent a unique swimming structure within the Crustacea.

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.

Austromegabalanus psittacus barnacle shell structure and proteoglycan localization and functionality.

Comparative analyzes of biomineralization models have being crucial for the understanding of the functional properties of biominerals and the elucidation of the processes through which biomacromolecules control the synthesis and structural organization of inorganic mineral-based biomaterials. Among calcium carbonate-containing bioceramics, egg, mollusk and echinoderm shells, and crustacean carapaces, have being fairly well characterized. However, Thoraceca barnacles, although being crustacea, showing molting cycle, build a quite stable and heavily mineralized shell that completely surround the animal, which is for life firmly cemented to the substratum. This makes barnacles an interesting model for studying processes of biomineralization. Here we studied the main microstructural and ultrastructural features of Austromegabalanus psittacus barnacle shell, characterize the occurrence of specific proteoglycans (keratan-, dermatan- and chondroitin-6-sulfate proteoglycans) in different soluble and insoluble organic fractions extracted from the shell, and tested them for their ability to crystallize calcium carbonate in vitro. Our results indicate that, in the barnacle model, proteoglycans are good candidates for the modification of the calcite crystal morphology, although the cooperative effect of some additional proteins in the shell could not be excluded.

Larval development of the pedunculate barnacles Octolasmis angulata Aurivillius 1894 and Octolasmis cor Aurivillius 1892 (Cirripedia: Thoracica: Poecilasmatidae) from the gills of the mud crab, Scylla tranquebarica Fabricius, 1798.

Detailed studies of larval development of Octolasmis angulata and Octolasmis cor are pivotal in understanding the larval morphological evolution as well as enhancing the functional ecology. Six planktotrophic naupliar stages and one non-feeding cyprid stage are documented in details for the first time for the two species of Octolasmis. Morphologically, the larvae of O. angulata and O. cor are similar in body size, setation patterns on the naupliar appendages, labrum, dorsal setae-pores, frontal horns, cyprid carapace, fronto-lateral gland pores, and lattice organs. Numbers of peculiarities were observed on the gnathobases of the antennae and mandible throughout the naupliar life-cycle. The setation pattern on the naupliar appendages are classified based on the segmentation on the naupliar appendages. The nauplius VI of both species undergoes a conspicuous change before metamorphosis into cyprid stage. The cyprid structures begin to form and modify beneath the naupliar body towards the end of stage VI. This study emphasises the importance of the pedunculate barnacle larval developmental studies not only to comprehend the larval morphological evolution but also to fill in the gaps in understanding the modification of the naupliar structures to adapt into the cyprid life-style.

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.

Trevathana noae, a new species of coral inhabiting barnacle (Cirripedia: Thoracica: Pyrgomatidae).

We describe a new species from the genus Trevathana Anderson, 1992, collected from the Cocos/Keeling Islands in the Indian Ocean. Trevathana noae sp. nov. is similar to other species of Trevathana by its external shell and opercular valve morphology. It is distinct from congeners in that the tergum has a distinct spur which lacks an internal tooth in adult specimens. A key to the known species of Trevathana is given. 

Morphology of the cement apparatus and the cement of the buoy barnacle Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae).

Barnacles produce a proteinaceous adhesive called cement to attach permanently to rocks or to other hard substrata. The stalked barnacle Dosima fascicularis is of special interest as it produces a large amount of foam-like cement that can be used as a float. The morphology of the cement apparatus and of the polymerized cement of this species is almost unknown. The current study aims at filling these gaps in our knowledge using light and electron microscopy as well as x-ray microtomography. The shape of the cement gland cells changes from round to ovoid during barnacle development. The cytoplasm of the gland cells, unlike that of some other barnacles, does not have distinct secretory and storage regions. The cement canals, which transport the cement from the gland cells to the base of the stalk, end at different positions in juvenile and mature animals. With increasing size of the cement float, the exit of the cement canals shift from the centrally positioned attachment disk of the vestigial antennules to more lateral positions on the stalk. The bubbles enclosed in the foam-like float are most likely filled with CO(2) that diffuses from the hemolymph into the cement canal system and from there into the cement.

Unusual adhesive production system in the barnacle Lepas anatifera: an ultrastructural and histochemical investigation.

Adhesives that are naturally produced by marine organisms are potential sources of inspiration in the search for medical adhesives. Investigations of barnacle adhesives are at an early stage but it is becoming obvious that barnacles utilize a unique adhesive system compared to other marine organisms. The current study examined the fine structure and chemistry of the glandular system that produces the adhesive of the barnacle Lepas anatifera. All components for the glue originated from large single-cell glands (70-180 µm). Staining (including immunostaining) showed that L-3,4-dihydroxyphenylalanine and phosphoserine were not present in the glue producing tissues, demonstrating that the molecular adhesion of barnacles differs from all other permanently gluing marine animals studied to date. The glandular tissue and adhesive secretion primarily consisted of slightly acidic proteins but also included some carbohydrate. Adhesive proteins were stored in cytoplasmic granules adjacent to an intracellular drainage canal (ICC); observations implicated both merocrine and apocrine mechanisms in the transport of the secretion from the cell cytoplasm to the ICC. Inside the ICC, the secretion was no longer contained within granules but was a flocculent material which became "clumped" as it traveled through the canal network. Hemocytes were not seen within the adhesive "apparatus" (comprising of the glue producing cells and drainage canals), nor was there any structural mechanism by which additions such as hemocytes could be made to the secretion. The unicellular adhesive gland in barnacles is distinct from multicellular adhesive systems observed in marine animals such as mussels and tubeworms. Because the various components are not physically separated in the apparatus, the barnacle adhesive system appears to utilize completely different and unknown mechanisms for maintaining the liquid state of the glue within the body, as well as unidentified mechanisms for the conversion of extruded glue into hard cement.

Metamorphosis in the cirripede crustacean Balanus amphitrite.

Stalked and acorn barnacles (Cirripedia Thoracica) have a complex life cycle that includes a free-swimming nauplius larva, a cypris larva and a permanently attached sessile juvenile and adult barnacle. The barnacle cyprid is among the most highly specialized of marine invertebrate larvae and its settlement biology has been intensively studied. By contrast, surprisingly few papers have dealt with the critical series of metamorphic events from cementation of the cyprid to the substratum until the appearance of a suspension feeding juvenile. This metamorphosis is both ontogenetically complex and critical to the survival of the barnacle. Here we use video microscopy to present a timeline and description of morphological events from settled cyprid to juvenile barnacle in the model species Balanus amphitrite, representing an important step towards both a broader understanding of the settlement ecology of this species and a platform for studying the factors that control its metamorphosis. Metamorphosis in B. amphitrite involves a complex sequence of events: cementation, epidermis separation from the cypris cuticle, degeneration of cypris musculature, rotation of the thorax inside the mantle cavity, building of the juvenile musculature, contraction of antennular muscles, raising of the body, shedding of the cypris cuticle, shell plate and basis formation and, possibly, a further moult to become a suspension feeding barnacle. We compare these events with developmental information from other barnacle species and discuss them in the framework of barnacle settlement ecology.

Construction and nanomechanical properties of the exoskeleton of the barnacle, Amphibalanus reticulatus.

Barnacles are some of the major inhabitants of intertidal zones and have calcite-based exoskeleton to anchor and armor their tissues. Structural characterization studies of the specie Ambhibalanus reticulatus were performed to understand the construction of the exoskeleton which forms a light-weight yet stiff structure. The parietal shell is constructed of six compartments to yield a truncated cone geometry, which is neatly fixed onto the basal shell that attaches the organism to the substrate surface. The connections among the different compartments happen through sutured edges and also have chemical interlocking to make the junctions impermeable. Also, the shell parts are furnished with hollow channels reducing the overall mass of the construction. The structure and functions of different parts of the exoskeleton are identified and outlined. Finally, the mechanical properties such as modulus, hardness and fracture toughness of the exoskeleton obtained by indentation techniques are discussed.

Interfacial morphology and nanomechanics of cement of the barnacle, Amphibalanus reticulatus on metallic and non-metallic substrata.

The barnacle exhibits a high degree of control over its attachment onto different types of solid surface. The structure and composition of barnacle cement have been reported previously, but mostly for barnacles growing on low surface energy materials. This article focuses on the strategies used by barnacles when they attach to engineering materials such as polymethylmethacrylate (PMMA), titanium (Ti) and stainless steel 316L (SS316L). Adhesion to these substrata is compared in terms of morphological structure, thickness and functional groups of the primary cement, the molting cycle and the nanomechanical properties of the cement. Structural characterization studies using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in conjunction with nanomechanical characterization and infrared spectroscopy (FTIR) are used to understand the differences in the adhesion of primary barnacle cement to the different substrata. The results provide new insights into understanding the mechanisms at work across the barnacle-substratum interface.

The structural, compositional and mechanical features of the calcite shell of the barnacle Tetraclita rufotincta.

The microstructure and chemical composition of the calcite shell of the sea barnacle Tetraclita rufotincta (Pilsbry, 1916) were investigated using microscopic and analytical methods. The barnacle shell was separated mechanically into its three substructural units: outer, interior, and inner layers. The organic matrices of these structural parts were further separated into soluble and insoluble constituents and their characteristic functional groups were studied by FTIR. Investigation of the mechanical properties of the interior mass of the shell reveals remarkable viscoelastic behavior. In general, the mechanical behavior of the shell is a function of its geometry as well as of the material, of which it is constructed. In the case of T. rufotincta, as calcite is a brittle material, the elastic behavior of the shell is apparently related to its micro- and macroarchitecture. The latter enables the shell to fulfill its primary function which is to protect the organism from a hostile environment and enables its survival. Our detailed identification of the similarities and differences between the various structural components of the shell in regard to the composition and properties of the organic component will hopefully throw light on the role of organic matrices in biomineralization processes.

Barnacle cement: an etchant for stainless steel 316L?

Localized corrosion of stainless steel beneath the barnacle-base is an unsolved issue for the marine industry. In this work, we clearly bring out for the first time the role of the barnacle cement in acting as an etchant, preferentially etching the grain boundaries, and initiating the corrosion process in stainless steel 316L. The investigations include structural characterization of the cement and corroded region, and also chemical characterization of the corrosion products generated beneath the barnacle-base. Structural characterization studies using scanning electron microscopy (SEM) reveals the morphological changes in the cement structure across the interface of the base-plate and the substrate, modification of the steel surface by the cement and the corrosion pattern beneath the barnacle-base. Fourier transform infrared spectroscopy (FTIR) of the corrosion products show that they are composed of mainly oxides of iron thereby implying that the corrosion is aerobic in nature. A model for the etching and corrosion mechanism is proposed based on our observations.

The monophyletic origin of a remarkable sexual system in akentrogonid rhizocephalan parasites: a molecular and larval structural study.

We use sequences from the nuclear ribosomal genes, 18S and 28S to analyze the phylogeny of the Rhizocephala Akentrogonida including two species, Clistosaccus paguri and Chthamalophilus delagei, that are critical for understanding rhizocephalan evolution but have not previously been part of a molecularly based study. In addition we use light and scanning electron microscopy to compare the cypris larvae of C. paguri, Sylon hippolytes and two species of the family Thompsoniidae, since this larval stage offers a suite of characters for analyzing the evolution of these otherwise highly reduced parasites. The Rhizocephala Akentrogonida form a monophyletic group nested within a paraphyletic "Kentrogonida". C. paguri and S. hippolytes are sistergroups confirming the monophyly of the Clistosaccidae that was originally based on similarities in the cypris larvae. We find numerous LM and SEM level similarities between the two species, many of which appear to be correlated with their specialized sexual system, where male cyprids use an antennule to implant cells into the virgin female parasite. Some of these traits are also found in cyprids of the thompsoniid species. We conclude that the special cypris morphology and the implantation of males by antennular penetration was present in the stem species to the Thompsoniidae and the Clistosaccidae and emphasize the power of larval characters in rhizocephalan systematics. C. delagei is a sister group to Boschmaella balani and the two are nested deep within the Akentrogonida. This confirms the monophyly of the Chthamalophilidae and falsifies the theory that C. delagei should represent the most primitive extant rhizocephalan. Instead, chthamalophilid rhizocephalans represent some of the most highly advanced members of the parasitic barnacles.

Nanoscale structures and mechanics of barnacle cement.

Polymerized barnacle glue was studied by atomic force microscopy (AFM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and chemical staining. Nanoscale structures exhibiting rod-shaped, globular and irregularly-shaped morphologies were observed in the bulk cement of the barnacle Amphibalanus amphitrite (=Balanus amphitrite) by AFM. SEM coupled with energy dispersive X-ray (EDX) provided chemical composition information, making evident the organic nature of the rod-shaped nanoscale structures. FTIR spectroscopy gave signatures of beta-sheet and random coil conformations. The mechanical properties of these nanoscale structures were also probed using force spectroscopy and indentation with AFM. Indentation data yielded higher elastic moduli for the rod-shaped structures when compared with the other structures in the bulk cement. Single molecule AFM force-extension curves on the matrix of the bulk cement often exhibited a periodic sawtooth-like profile, observed in both the extend and retract portions of the force curve. Rod-shaped structures stained with amyloid protein-selective dyes (Congo red and thioflavin-T) revealed that about 5% of the bulk cement were amyloids. A dominant 100 kDa cement protein was found to be mechanically agile, using repeating hydrophobic structures that apparently associate within the same protein or with neighbors, creating toughness on the 1-100 nm length scale.

Cypris morphology in the barnacles Ibla and Paralepas (Crustacea: Cirripedia Thoracica) implications for cirripede evolution.

We used scanning electron microscopy (SEM) to describe cypris morphology in species of the barnacles Ibla and Paralepas, both of which are pivotal in understanding cirripede evolution. In Ibla, we also studied late naupliar stages with video and SEM. Special emphasis was put on the lattice organs, the antennules and the thorax and telson. In Paralepas we had settled specimens only and could therefore only investigate the carapace with the lattice organs. Cyprids of Ibla quadrivalvis and Paralepas dannevigi have five sets of lattice organs, grouped as two anterior and three posterior pairs. The organs are of the pore-field type and the terminal pore is situated anteriorly in the first pair, just as in the Rhizocephala and the Thoracica. In Ibla the armament of antennular sensilla resembles that found in the Thoracica but differs from the Rhizocephala. The absence of setules on the A and B setae sited terminally on the fourth antennular segment is a similarity with the Acrothoracica. The attachment disc is angled rather than facing distally and is encircled by a low cuticular velum. The thoracopods have two-segmented endopods and exopods as in the Thoracica, but the number, shape, and position of thoracopodal setae differ somewhat from other species of that superorder. Both Ibla and Paralepas cyprids have a deeply cleaved telson, but no independent abdominal part. In cypris morphology, Ibla and Paralepas show several synapomorphies with the clade comprising Rhizocephala and Thoracica and there are no specific apomorphies with either the Acrothoracica, the Rhizocephala or any particular subgroup within the Thoracica. This is in agreement with recent molecular evidence that Ibla (Ibliformes) is the sister taxon to all other Thoracica and the ibliforms therefore become the outgroup of choice for studying character evolution within the superorder. Paralepas, and other pedunculated barnacles without shell plates, are apparently not primitive but are secondarily evolved and nested within the Thoracica.

Marine biofouling field tests, settlement assay and footprint micromorphology of cyprid larvae of Balanus amphitrite on model surfaces.

Atomic force microscopy (AFM), laboratory settlement assays and field tests were used to correlate cyprid footprint (FP) morphology with the behaviour of cyprids on different substrata. AFM imaging under laboratory conditions revealed more porous and larger FPs on glass exposing a CH3-surface than on aminosilane functionalised (NH2-) surfaces. The secreted FP volume was found to be similar on both substrata (2.1-2.6 microm(3)). Laboratory settlement assays and marine field tests were performed on three substrata, viz. untreated clean glass, NH2-glass, and CH3-glass. The results distinguished settlement preferences for NH2-glass and untreated glass over CH3-terminated surfaces, suggesting that cyprids favour settling on hydrophilic over hydrophobic surfaces. On combining observations from different length scales, it is speculated that the confined FP size on NH2-glass may induce a higher concentration of the settlement inducing protein complex. Settlement may be further facilitated by a stronger adherence of FP adhesives to the NH2-surface via Coulombic interactions.

Three-dimensional reconstruction of the naupliar musculature and a scanning electron microscopy atlas of nauplius development of Balanus improvisus (Crustacea: Cirripedia: Thoracica).

An atlas of the naupliar development of the cirripede Balanus improvisus Darwin, 1854 using scanning electron microscopy (SEM) is provided. Existing spikes on the hindbody increase in number with each moult and are an applicable character for identification of the different nauplius stages, as is the setation pattern of the first antennae. The naupliar musculature of B. improvisus was stained with phalloidin to visualise F-actin, followed by analysis using confocal laser scanning microscopy (CLSM) with subsequent application of 3D imaging software. The larval musculature is already fully established in the first nauplius stage and remains largely unchanged during all the six nauplius stages. The musculature associated with the feeding apparatus is highly elaborated and the labrum possesses lateral muscles and distal F-actin-positive structures. The alimentary tract is entirely surrounded by circular muscles. The extrinsic limb musculature comprises muscles originating from the dorsal and the ventral sides of the head shield, respectively. The hindbody shows very prominent postero-lateral muscles that insert on the dorso-lateral side of the head shield and bend towards ventro-posterior. We conclude that the key features of the naupliar gross anatomy and muscular architecture of B. improvisus are important characters for phylogenetic inferences if analysed in a comparative evolutionary framework.

Hepatopancreas alteration of the blue crab Callinectes sapidus by the rhizocephalan barnacle Loxothylacus texanus.

A histological study of the hepatopancreas of the blue crab Callinectes sapidus parasitized by the rhizocephalan barnacle Loxothylacus texanus was conducted to explore if the degree of development of the parasite's rootlet system was correlated to its maturation process as seen by external characters of its reproductive body or externa. Four types of crabs were examined: control, with virgin and mature externa, and scarred. A clear progression with an increase in number and size of the parasite's rootlets in the hosts' hepatopancreas can be seen. Although the hepatopancreatic tubules remain functional, the hepatopancreas appears as a loose structure, completely infiltrated with L. texanus rootlets, in advanced stages of the parasitism.