Cypris Larvae Research Articles

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.

Effect of chlorination on barnacle larval stages: Implications for biofouling control and environmental impact

Continuous low-dose chlorination (CLDC, 0.2 ± 0.1 mg l−1) is practised to control mussel settlement in industrial cooling systems. However, its effect on larval barnacles is poorly understood. Experiments were carried out with larvae of the barnacle Amphibalanus reticulatus to assess: 1) chlorine toxicity to discrete naupliar stages, 2) effect of transient chlorine exposure of nauplii on subsequent larval development and 3) effect of chlorine on cypris settlement and metamorphosis. Individual naupliar stages showed different levels of sensitivity to chlorine exposures with the stage II nauplii being the most susceptible to in-use levels (0.2 ± 0.1 mg l−1) of chlorination. Short term exposures (3–20 min) to 0.2 mg l−1chlorine did not prevent larval settlement. A significant percentage of treated cyprid larvae (non-feeding stage) survived, settled and metamorphosed to juveniles. A fraction of the chlorine-treated cypris larvae precociously metamorphosed into juveniles, bypassing cementation to substratum. About half of the pre-competent larvae (naupliar stages) exposed to in-use levels of chlorine failed to reach cypris stage, indicating that CLDC can impact barnacle larval development in the sea. In barnacle dominated systems, use of alternative control strategies/biocides may be required.

Biochemical Analysis of Adhesives Produced by the Cypris Larvae of Barnacle Amphibalanus amphitrite

Barnacles are the dominant organisms in the biofouling communities throughout the world seas and attain considerable ecological and economic significance. In the present study, cement produced by the cypris larva of the barnacle Amphibalanus amphitrite was analyzed to understand the biochemical composition of the adhesive material. Protein content showed higher values (408.13 μg mg−1) than lipids and carbohydrates. The thin-layer chromatography analysis showed a single distinct spot indicating a carbohydrate group in the cement. SDS-PAGE analysis of the adhesives revealed protein bands between the molecular weight range of 20.1–43 kDa and also 66 kDa. The cement was also analyzed using FT-IR and indicated the presence of peak related to non-amyloid beta sheets. In conclusion, the cement isolated from the barnacle larvae contains protein as major components with the possibility of the presence of non-amyloid globular proteins.

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.

STUDIEN AN KARIBISCHEN CIRRIPEDIEN I. ERGANZENDE BESCHREIBUNG VON SCALPELLUM REGINA PILSBRY, 1907

A supplementary description of Scalpellum regina is given, based on the morphology of mouth parts, cirri, abdominal appendages and penis. Also for the first time are described and illustrated the cypris larva and complemental males of this species. There are given some remarks on the substrata of the specimens found in Colombian waters, and briefly discussed the intrageneric relationships of Scalpellum.

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.

In vivo and in situ synchrotron radiation-based μ-XRF reveals elemental distributions during the early attachment phase of barnacle larvae and juvenile barnacles.

Barnacles are able to establish stable surface contacts and adhere underwater. While the composition of adult barnacle cement has been intensively studied, far less is known about the composition of the cement of the settlement-stage cypris larva. The main challenge in studying the adhesives used by these larvae is the small quantity of material available for analysis, being on the order of nanograms. In this work, we applied, for the first time, synchrotron radiation-based μ-X-ray fluorescence analysis (SR-μ-XRF) for in vivo and in situ analysis of young barnacles and barnacle cyprids. To obtain biologically relevant information relating to the body tissues, adhesives, and shell of the organisms, an in situ sample environment was developed to allow direct microprobe investigation of hydrated specimens without pretreatment of the samples. In 8-day-old juvenile barnacles (Balanus improvisus), the junctions between the six plates forming the shell wall showed elevated concentrations of calcium, potassium, bromine, strontium, and manganese. Confocal measurements allowed elemental characterization of the adhesive interface of recently attached cyprids (Balanus amphitrite), and substantiated the accumulation of bromine both at the point of initial attachment as well as within the cyprid carapace. In situ measurements of the cyprid cement established the presence of bromine, chlorine, iodine, sulfur, copper, iron, zinc, selenium, and nickel for both species. The previously unrecognized presence of bromine, iron, and selenium in the cyprid permanent adhesive will hopefully inspire further biochemical investigations of the function of these substances.

Sex determination in the androdioecious barnacleScalpellum scalpellum(Crustacea: Cirripedia)

How androdioecy (coexistence of hermaphrodites and males) is maintained is still poorly understood. Therefore, sex determination was studied in the androdioecious barnacle Scalpellum scalpellum L. First, 247 cypris larvae from seven broods were investigated for sexual dimorphism in larval morphology and found to be all identical. Second, experiments with cyprids showed that males and hermaphrodites differ distinctly in morphology as soon as 4–5 days after settlement. Third, 14 252 cyprids were allowed to settle on the bottom of their culture cages, and all surviving larvae developed into hermaphrodites and none into dwarf males. Fourth, larvae settled in hermaphrodite receptacles (i.e. future males) were removed at increasing intervals after settlement to study if the male and hermaphrodite sexual expressions are fixed or plastic. All larvae became dwarf males if allowed to stay there for more than 8 h after settlement. But if removed within 3 h after settlement, half of them developed into hermaphrodites. We conclude that an environmental sex determination mechanism operates in S. scalpellum. Together with a 1:1 hermaphrodite/male ratio observed in previously reported experiments offering a free choice of settlement, we suggest that all larvae are potential hermaphrodites, but only 50% can settle in hermaphrodite receptacles and yield males.

Barnacle settlement on rocky shores: Substratum preference and epibiosis on mussels

Understanding the dynamics of epibiosis is fundamental to understanding the role of biological interactions in the functioning of marine ecosystems. At many coastal sites, barnacles are abundant as epibionts on the shells of mussels. As they approach the shore, cypris larvae explore the environment, actively seeking suitable settlement sites and rejecting unsuitable substrata. Suitability involves micro-features of the surface, including its texture and contours, chemical signals and the presence of conspecifics. To investigate whether epibiosis on mussel shells indicates a preference for this substratum on the part of the barnacle Chthamalus dentatus, artificial plates were deployed at two rocky shore sites on the south coast of South Africa. These plates offered settling cyprids four habitat choices: a live mussel, the shell of a recently dead mussel, a fine resolution resin replica of a mussel shell to mimic architectural micro-surface, without the chemical characteristics of natural shells and a rock mimic, a surface covered in a film of hard plastic that resembled natural rock surface as closely as possible. Settling surfaces were photographed monthly, and new barnacles were counted and allocated to size classes, so that survival could be estimated over a period of several months. Barnacles showed a clear preference for the rock mimic surfaces, which on average supported double the number of settlers found on mussel replicas. These in turn were significantly higher than the numbers on live and dead mussel shell surfaces, implying that there are features of both dead and live mussel shells that deter barnacle settlement. In contrast, final abundances of adults showed no significant effect of settling surface. The results suggest that during settlement, cyprids avoid chemical cues from mussel shells that persist even after the death of the mussel. This suggests that the high numbers of barnacles often found on the surface of live mussels could be due to saturation of substratum.

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.

Degradation kinetics of a potent antifouling agent, butenolide, under various environmental conditions

Here, we investigated the degradation kinetics of butenolide, a promising antifouling compound, under various environmental conditions. The active ingredient of the commercial antifoulant SeaNine 211, 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT), was used as positive control. The results showed that the degradation rate increased with increasing temperature. Half-lives of butenolide at 4°C, 25°C and 40°C were>64d, 30.5d and 3.9d, respectively. Similar half-lives were recorded for DCOIT: >64d at 4°C, 27.9d at 25°C and 4.5d at 40°C. Exposure to sunlight accelerated the degradation of both butenolide and DCOIT. The photolysis half-lives of butenolide and DCOIT were 5.7d and 6.8d, respectively, compared with 9.7d and 14.4d for the dark control. Biodegradation led to the fastest rate of butenolide removal from natural seawater, with a half-life of 0.5d, while no obvious degradation was observed for DCOIT after incubation for 4d. The biodegradative ability of natural seawater for butenolide was attributed mainly to marine bacteria. During the degradation of butenolide and DCOIT, a gradual decrease in antifouling activity was observed, as indicated by the increased settlement percentage of cypris larvae from barnacle Balanus amphitrite. Besides, increased cell growth of marine diatom Skeletonema costatum demonstrated that the toxicity of seawater decreased gradually without generation of more toxic by-products. Overall, rapid degradation of butenolide in natural seawater supported its claim as a promising candidate for commercial antifouling industry.

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.

The origin of barnacles (Thecostraca, Cirripedia)

I claim that hybridogenesis has played a vital part in the evolution of the Thoracica, Acrothoracica, Rhizocephala and Facetotecta. Hybridogenesis is the generation of new life forms and new life histories by sexual hybridization. It includes larval transfer, which explains the origins of all larvae, and component transfer, which explains the origins of cnidarians, lophophorates and thoracicans. I propose that adult thoracicans are descended from a hybrid between a crustacean and a non-arthropod, the genomes of which are expressed together. They are concurrent chimeras. Adult acrothoracicans are equivalent to the crustacean part of thoracicans. Nauplius larvae were added to the life histories of thoracicans and some acrothoracicans by later hybridizations. Adult rhizocephalans and juvenile facetotectans have no arthropod characteristics, and I claim that both these taxa acquired nauplius and cypris larvae by hybrid transfer. Extreme differences between the mitochondrial genomes of a sacculinid rhizocephalan and thoracicans are consistent with this view of rhizocephalan evolution. The Rhizocephala and Facetotecta are not members of the Cirripedia or the Arthropoda.

Slippery Liquid-Infused Porous Surfaces Showing Marine Antibiofouling Properties

Marine biofouling is a longstanding problem because of the constant challenges placed by various fouling species and increasingly restricted environmental regulations for antifouling coatings. Novel nonbiocidal strategies to control biofouling will necessitate a multifunctional approach to coating design. Here we show that slippery liquid-infused porous surfaces (SLIPSs) provide another possible strategy to obtaining promising antifouling coatings. Microporous butyl methacrylate-ethylene dimethacrylate (BMA-EDMA) surfaces are prepared via UV-initiated free-radical polymerization. Subsequent infusion of fluorocarbon lubricants (Krytox103, Krytox100, and Fluorinert FC-70) into the porous microtexture results in liquid-repellent slippery surfaces. To study the interaction with marine fouling organisms, settlement of zoospores of the alga Ulva linza and cypris larvae of the barnacle Balanus amphitrite is tested in laboratory assays. BMA-EDMA surfaces infused with Krytox103 and Krytox100 exhibit remarkable inhibition of settlement (attachment) of both spores and cyprids to a level comparable to that of a poly(ethylene glycol) (PEG)-terminated self-assembled monolayer. In addition, the adhesion strength of sporelings (young plants) of U. linza is reduced for BMA-EDMA surfaces infused with Krytox103 and Krytox100 compared to pristine (noninfused) BMA-EDMA and BMA-EDMA infused with Fluorinert FC-70. Immersion tests suggest a correlation between the stability of slippery coatings in artificial seawater and fouling resistance efficacy. The results indicate great potential for the application of this concept in fouling-resistant marine coatings.

Effects of nano carbon black and single-layer graphene oxide on settlement, survival and swimming behaviour of Amphibalanus amphitrite larvae

The effects of two carbon-based nanomaterials, nano-sized carbon black (nCB), and single-layer graphene oxide (GO) on settlement of Amphibalanus amphitrite (Cirripedia, Crustacea) cypris larvae (cyprids) were assessed after 24, 48, and 72 h of exposure. Additionally, the effects of these nanomaterials on the mortality and swimming behaviour of the nauplius larvae (nauplii) of the same organism were determined after 24 and 48 h. The data indicate that nCB is more effective as a potential antisettlement agent than single-layer GO; moreover, nCB did not show any adverse effects on the larvae. The swimming behaviour of II stage nauplii of A. amphitrite exposed to a suspension of nCB was inhibited only at very high nCB concentrations (≥0.5 mg/mL). Single-layer GO, on the contrary, showed lower antisettlement effects and was more active in altering the survival and inhibiting the swimming behaviour of the nauplii. An indication of the toxic or non-toxic mechanisms of the antisettlement properties of both of these nanomaterials is provided by the reversibility of the antisettlement activity. In conclusion, we propose nCB as an innovative antifouling nanomaterial that shows low toxicity towards the model organism (crustaceans) used in this study.

Modeling of the invasion and development of the pelagic communities of fouling organisms in the ocean

The results of the modeling of the origin and development of the oceanic fouling biocenoses in the pelagic zone are presented. The models are relatively simple, but they are characterized by major peculiarities of the natural communities. The fouling communities are model objects to study the appearance and development of biological systems, invasions of hydrobionts, application of the bioindicator species, etc. The presented models take into account the most important interactions of the main fouling species (Lepadidae crustaceans are the dominating species) in typical regions and depths for such invasion and at the favorable abiotic environmental factors that are comparatively stable. The interaction between the meroplanktonic larvae of Lepas and Conchoderma (Lepadidae), represented by C. virgatum, L. anatifera, L. anserifera, and L. hillii, and the most widespread predators (crabs Planes minutus and the fish species) is modeled as the system of differential equations. In particular, the model covers the dynamics of colonies of fouling species, the “attractiveness” of these colonies to other fouling species, and the feeding rates of the predators consuming both the fouling species and associated fauna. The computing experiments showed that the population density changed markedly in the different age groups of Lepas and Conchoderma. The predator stress on Conchoderma was significantly higher than that on Lepas. The “attractiveness” of the biofouling organisms to cypris larvae and predators keeping near the substrate has a major impact on their density dynamics. The models showed that the crabs and especially fish had the strongest effect on the biocenoses of oceanic biofouling communities, limiting the population density of fouling species. When the presence of fish was excluded from the model, the abundance of Conchoderma increased rapidly and became similar to the abundance of Lepas. The model showed that the most important factors limiting the oceanic fouling are the settling rate of Lepadidae larvae to the substrate, “attractiveness” of the fouling organisms to predators, and the consumption of these animals by the predators. The models are in good correlation with experimental data of oceanographic buoy areas. Usage of the mathematical models for forecasting and assessing the invasion rates of biofouling organisms and the further development of their biocenoses is very promising and cannot be totally replaced by the other research methods. Analogous models can be developed for other communities and marine environments, for example, for the forecasting of hydrobiont invasions into the marine and freshwater bodies of Russia.

Scanning electron microscopy of the cypris larvae ofCapitulum mitella(Cirripedia: Thoracica: Scalpellomorpha)

Adult specimens ofCapitulum mitellawere collected in August 2011 in the intertidal zone of Dinghai, Fuzhou, Fujian, China (26°16′N 119°48′E). The morphology of the cypris larva reared under our conditions was determined using scanning electron microscopy (SEM). Special emphasis was given to the carapace, lattice organs, antennules, thoracopods and furcal rami. The whole carapace surface is sculptured by slender ridges demarcating rectangular or irregular polygonal areas with very fine pores. The cyprid possesses five pairs of lattice organs, arranged as two anterior and three posterior pairs. The second segment carries a preaxial seta 2 and a postaxial seta 2. The third segment carries a postaxial seta 3 outside the attachment disc, whereas a postaxial disc seta, an axial disc seta and two radial disc setae are located inside the attachment disc. The attachment disc is somewhat bell shaped. A series of slender cuticular flaps forms a distinct ‘velum’ around the base of the disc. The fourth segment carries four subterminal setae and five terminal setae. The cyprid bears six pairs of biramous natatory thoracopods consisting of a protopod (coxa + basis), a two-segmented exopod and a two-segmented endopod. The cyprid possesses a rudimentary abdomen and an almost completely cleaved telson with a pair of one-segmented furcal rami. The morphology and setation of the antennules ofC. mitellaresemble those ofBalanus amphitrite(=Amphibalanus amphitrite) andMegabalanus rosa, but differ in some morphological details.

Analysis of the Behaviours Mediating Barnacle Cyprid Reversible Adhesion

When exploring immersed surfaces the cypris larvae of barnacles employ a tenacious and rapidly reversible adhesion mechanism to facilitate their characteristic ‘walking’ behaviour. Although of direct relevance to the fields of marine biofouling and bio-inspired adhesive development, the mechanism of temporary adhesion in cyprids remains poorly understood. Cyprids secrete deposits of a proteinaceous substance during surface attachment and these are often visible as ‘footprints’ on previously explored surfaces. The attachment structures, the antennular discs, of cyprids also present a complex morphology reminiscent of both the hairy appendages used by some terrestrial invertebrates for temporary adhesion and a classic ‘suction cup’. Despite the numerous analytical approaches so-far employed, it has not been possible to resolve conclusively the respective contributions of viscoelastic adhesion via the proteinaceous ‘temporary adhesive’, ‘dry’ adhesion via the cuticular villi present on the disc and the behavioural contribution by the organism. In this study, high-speed photography was used for the first time to capture the behaviour of cyprids at the instant of temporary attachment and detachment. Attachment is facilitated by a constantly sticky disc surface – presumably due to the presence of the proteinaceous temporary adhesive. The tenacity of the resulting bond, however, is mediated behaviourally. For weak attachment the disc is constantly moved on the surface, whereas for a strong attachment the disc is spread out on the surface. Voluntary detachment is by force, requiring twisting or peeling of the bond – seemingly without any more subtle detachment behaviours. Micro-bubbles were observed at the adhesive interface as the cyprid detached, possibly an adaptation for energy dissipation. These observations will allow future work to focus more specifically on the cyprid temporary adhesive proteins, which appear to be fundamental to adhesion, inherently sticky and exquisitely adapted for reversible adhesion underwater.

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

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

Morphometric and molecular identification of individual barnacle cyprids from wild plankton: an approach to detecting fouling and invasive barnacle species

The present study used DNA barcodes to identify individual cyprids to species. This enables accurate quantification of larvae of potential fouling species in the plankton. In addition, it explains the settlement patterns of barnacles and serves as an early warning system of unwanted immigrant species. Sequences from a total of 540 individual cypris larvae from Taiwanese waters formed 36 monophyletic clades (species) in a phylogenetic tree. Of these clades, 26 were identified to species, but 10 unknown monophyletic clades represented non-native species. Cyprids of the invasive barnacle, Megabalanus cocopoma, were identified. Multivariate analysis of antennular morphometric characters revealed three significant clusters in a nMDS plot, viz. a bell-shaped attachment organ (most species), a shoe-shaped attachment organ (some species), and a spear-shaped attachment organ (coral barnacles only). These differences in attachment organ structure indicate that antennular structures interact directly with the diverse substrata involved in cirripede settlement.