Pressure cycling technology for challenging proteomic sample processing: application to barnacle adhesive.

Successful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730.

Incorporation of silicone oil into elastomers enhances barnacle detachment by active surface strain.

Silicone-oil additives are often used in fouling-release silicone coatings to reduce the adhesion strength of barnacles and other biofouling organisms. This study follows on from a recently reported active approach to detach barnacles, which was based on the surface strain of elastomeric materials, by investigating a new, dual-action approach to barnacle detachment using Ecoflex®-based elastomers incorporated with poly(dimethylsiloxane)-based oil additives. The experimental results support the hypothesis that silicone-oil additives reduce the amount of substratum strain required to detach barnacles. The study also de-coupled the two effects of silicone oils (ie surface-activity and alteration of the bulk modulus) and examined their contributions in reducing barnacle adhesion strength. Further, a finite element model based on fracture mechanics was employed to qualitatively understand the effects of surface strain and substratum modulus on barnacle adhesion strength. The study demonstrates that dynamic substratum deformation of elastomers with silicone-oil additives provides a bifunctional approach towards management of biofouling by barnacles.

How do coral barnacles start their life in their hosts?

Coral-associated invertebrates are the most significant contributors to the diversity of reef ecosystems, but no studies have examined how larvae manage to settle and grow in their coral hosts. Video recordings were used to document this process in the coral barnacle Darwiniella angularis associated with the coral Cyphastrea chalcidicum Settlement and metamorphosis in feeding juveniles lasted 8-11 days and comprised six phases. The settling cyprid starts by poking its antennules into the tissue of the prospective host (I: probing stage). The coral releases digestive filaments for defence, but tolerating such attack the cyprid penetrates further (II: battling stage). Ecdysis is completed 2 days after settlement (III: carapace detachment). The barnacle becomes embedded deep in the coral tissue while completing metamorphosis between 4 and 6 days (IV: embedding stage), but reappears as a feeding juvenile 8-11 days after settlement (V: emerging stage; VI: feeding stage). Cyprids preferably settle in areas between the coral polyps, where they have a much higher survival rate than on the polyp surfaces.

Characterization of biofilm formation in natural water subjected to low-frequency electromagnetic fields.

Electromagnetic field (EMF) treatment has proven to be effective against mineral scaling in water systems. Therefore, it should be assessed for the treatment of other deposits such as biofilms. In this study, a commercial device producing low-frequency EMF (1-10 kHz) was applied to a reactor fed with natural water for 45 days. The treatment promoted the concentration of microorganisms in suspension and limited the amount of sessile microorganisms in the biofilm, as determined by the measurement of total DNA, qPCR and microscopy. The structure of the bacterial community was assessed by t-RFLP and pyrosequencing analysis. The results showed that EMF treatment affected both planktonic and sessile community composition. EMFs were responsible for a shift in classes of Proteobacteria during development of the biofilm. It may be speculated that the EMF treatment affected particle solubility and/or microorganism hydration. This study indicated that EMFs modulated biofilm formation in natural water.

Relationship between metal and polybrominated diphenyl ether (PBDE) body burden and health risks in the barnacle Balanus amphitrite.

In the present study, we employed the widespread and gregarious barnacle species Balanus amphitrite in a biomonitoring program to evaluate coastal pollution around three piers (i.e., Tso Wo Hang, Sai Kung and Hebe Haven) in Hong Kong. An integrated approach was used herein, combining both the chemical determination of contaminant concentrations, including metals and polybrominated diphenyl ethers (PBDEs), and a suite of biological responses across the entire barnacle lifecycle (i.e., adult, nauplius, cyprid and juvenile). The analytical results revealed a distinct geographical distribution of metals and PBDEs. Adult physiological processes and larval behaviors varied significantly among the three piers. Furthermore, a correlation analysis demonstrated a specific suite of biological responses towards metal and PBDE exposure, likely resulting from their distinct modes of action. Overall, the results of this study indicated that the combination of chemical and biological tests provided an integrated measure for the comprehensive assessment of marine pollution.

Bioactive diphenyl ether derivatives from a gorgonian-derived fungus Talaromyces sp.

Three new diphenyl ether derivatives, talaromycins A-C (1-3, resp.), together with six known analogs, 4-9, were isolated from a gorgonian-derived fungus, Talaromyces sp. The structures of the new compounds were determined by analysis of extensive NMR spectroscopic data. All of the isolated metabolites, 1-9, were evaluated for their cytotoxic and antifouling activities. Compound 4 exhibited pronounced cytotoxicity against the tested human cell lines with the IC50 values ranging from 4.3 to 9.8 µM. Compounds 3, 5, 8, and 9 showed potent antifouling activities against the larval settlement of the barnacle Balanus amphitrite with the EC50 values ranging from 2.2 to 4.8 µg/ml.

Tea stains-inspired initiator primer for surface grafting of antifouling and antimicrobial polymer brush coatings.

Inspired by tea stains, plant polyphenolic tannic acid (TA) was beneficially employed as the primer anchor for functional polymer brushes. The brominated TA (TABr) initiator primer was synthesized by partial modification of TA with alkyl bromide functionalities. TABr with trihydroxyphenyl moieties can readily anchor on a wide range of substrates, including metal, metal oxide, polymer, glass, and silicon. Concomitantly, the alkyl bromide terminals serve as initiation sites for atom transfer radical polymerization (ATRP). Cationic [2-(methacryloyloxy)ethyl]trimethylammonium chloride (META) and zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) and N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine (SBMA) were graft-polymerized from the TABr-anchored stainless steel (SS) surface. The cationic polymer brushes on the modified surfaces are bactericidal, while the zwitterionic coatings exhibit resistance against bacterial adhesion. In addition, microalgal attachment (microfouling) and barnacle cyprid settlement (macrofouling) on the functional polymer-grafted surfaces were significantly reduced, in comparison to the pristine SS surface. Thus, the bifunctional TABr initiator primer provides a unique surface anchor for the preparation of functional polymer brushes for inhibiting both microfouling and macrofouling.

Sulfobetaine-based polymer brushes in marine environment: is there an effect of the polymerizable group on the antifouling performance?

Three different zwitterionic polymer brush coatings for marine biofouling control were prepared by surface-initiated atom transfer radical polymerization (ATRP) of sulfobetaine-based monomers including methacrylamide (SBMAm), vinylbenzene (SBVB) and vinylimidazolium (SBVI). None of these brush systems have been assessed regarding marine antifouling performance. Antifouling tests performed indicate that surfaces featuring these three brush systems substantially reduce the adhesion of the marine microalgae, Amphora coffeaeformis, and the settlement of cyprid larvae of the barnacle, Amphibalanus amphitrite, in a similar way, displaying comparable performance. Thus, it appears that the chemical structure of the polymerizable group has no substantial influence on marine antifouling performance.

Molecular characterization of the α-subunit of Na⁺/K⁺ ATPase from the euryhaline barnacle Balanus improvisus reveals multiple genes and differential expression of alternative splice variants.

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na⁺/K⁺ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.

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.

Poly(dimethyl siloxane) (PDMS) network blends of amphiphilic acrylic copolymers with poly(ethylene glycol)-fluoroalkyl side chains for fouling-release coatings. II. Laboratory assays and field immersion trials.

Amphiphilic copolymers containing different amounts of poly(ethylene glycol)-fluoroalkyl acrylate and polysiloxane methacrylate units were blended with a poly(dimethyl siloxane) (PDMS) matrix in different proportions to investigate the effect of both copolymer composition and loading on the biological performance of the coatings. Laboratory bioassays revealed optimal compositions for the release of sporelings of Ulva linza, and the settlement of cypris larvae of Balanus amphitrite. The best-performing coatings were subjected to field immersion tests. Experimental coatings containing copolymer showed significantly reduced levels of hard fouling compared to the control coatings (PDMS without copolymer), their performance being equivalent to a coating based on Intersleek 700™. XPS analysis showed that only small amounts of fluorine at the coating surface were sufficient for good antifouling/fouling-release properties. AFM analyses of coatings under immersion showed that the presence of a regular surface structure with nanosized domains correlated with biological performance.

Corrosion behaviour and biofouling characteristics of structural steel in the coastal waters of the Gulf of Mannar (Bay of Bengal), India.

The corrosion behaviour and biofouling characteristics of structural steel coupons at three different locations in the Gulf of Mannar were studied over a period of 2 years. Oyster fouling was predominant at Tuticorin open sea, while barnacle fouling was more pronounced at Mandapam and Tuticorin harbour. Among the three locations, Tuticorin open sea showed a markedly higher biomass, particularly after 12 and 18 months. The extent of crevice corrosion caused by hard foulers was more pronounced at Tuticorin harbour when compared to that at the other two locations. The corrosion rate of the structural steel coupons for 24 months was in the order, Mandapam > Tuticorin harbor > Tuticorin open sea. The loss in tensile strength at 12 and 24 months was in the order, Tuticorin open sea > Tuticorin harbor > Mandapam. The corrosion behaviour of the structural steel coupons was strongly influenced by the variations in the biofouling assemblage at the three different coastal locations.

Inhibition of barnacle cyprid settlement using low frequency and intensity ultrasound.

Low frequency, low intensity ultrasound was demonstrated as an effective inhibitor of barnacle cyprid settlement. When the same substratum vibration amplitude (10.05 nm) and acoustic pressure (5 kPa) were applied, ultrasound at a frequency of 23 kHz significantly reduced cyprid settlement. The mechanism appeared to differ from the ultrasonic cavitation induced inhibition previously reported as no increased mortality was observed, and no change in the exploratory behaviour of cyprids was observed when they were exposed to this continuous ultrasonic irradiation regime. The application of ultrasound treatment in an intermittent mode of '5 min on and 20 min off' at 20-25 kHz and at the low intensity of 5 kPa produced the same effect as the continuous application of 23 kHz. This energy efficient approach to the use of low frequency, low intensity ultrasound may present a promising and efficient strategy regarding irradiation treatment for antifouling applications.

Investigation of the role of hydrophilic chain length in amphiphilic perfluoropolyether/poly(ethylene glycol) networks: towards high-performance antifouling coatings.

The facile preparation of amphiphilic network coatings having a hydrophobic dimethacryloxy-functionalized perfluoropolyether (PFPE-DMA; M(w) = 1500 g mol(-1)) crosslinked with hydrophilic monomethacryloxy functionalized poly(ethylene glycol) macromonomers (PEG-MA; M(w) = 300, 475, 1100 g mol(-1)), intended as non-toxic high-performance marine coatings exhibiting antifouling characteristics is demonstrated. The PFPE-DMA was found to be miscible with the PEG-MA. Photo-cured blends of these materials containing 10 wt% of PEG-MA oligomers did not swell significantly in water. PFPE-DMA crosslinked with the highest molecular weight PEG oligomer (ie PEG1100) deterred settlement (attachment) of algal cells and cypris larvae of barnacles compared to a PFPE control coating. Dynamic mechanical analysis of these networks revealed a flexible material. Preferential segregation of the PEG segments at the polymer/air interface resulted in enhanced antifouling performance. The cured amphiphilic PFPE/PEG films showed decreased advancing and receding contact angles with increasing PEG chain length. In particular, the PFPE/PEG1100 network had a much lower advancing contact angle than static contact angle, suggesting that the PEG1100 segments diffuse to the polymer/water interface quickly. The preferential interfacial aggregation of the larger PEG segments enables the coating surface to have a substantially enhanced resistance to settlement of spores of the green seaweed Ulva, cells of the diatom Navicula and cypris larvae of the barnacle Balanus amphitrite as well as low adhesion of sporelings (young plants) of Ulva, adhesion being lower than to a polydimethyl elastomer, Silastic T2.

Modulation of barnacle (Balanus amphitrite Darwin) cyprid settlement behavior by sulfobetaine and carboxybetaine methacrylate polymer coatings.

Zwitterionic polymers such as poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA) have demonstrated impressive fouling-resistance against proteins and mammalian cells. In this paper, the effects of these surface chemistries on the settlement and behavior of an ubiquitous fouling organism, the cypris larva of the barnacle Balanus amphitrite (=Amphibalanus amphitrite), were studied in the laboratory. Conventional settlement assays and behavioral analysis of cyprids using Noldus Ethovision 3.1 demonstrated significant differences in settlement and behavior on different surfaces. Cyprids did not settle on the polySBMA or polyCBMA surfaces over the course of the assay, whereas settlement on glass occurred within expected limits. Individual components of cyprid behavior were shown to differ significantly between glass, polySBMA and polyCBMA. Cyprids also responded differently to the two zwitterionic surfaces. On polySBMA, cyprids were unwilling or unable to settle, whereas on polyCBMA cyprids did not attempt exploration and left the surface quickly. In neither case was toxicity observed. It is concluded that a zwitterionic approach to fouling-resistant surface development has considerable potential in marine applications.

Natural antifoulants from the marine cyanobacterium Lyngbya majuscula.

Filamentous benthic marine cyanobacteria are a prolific source of structurally unique bioactive secondary metabolites. A total of 12 secondary metabolites, belonging to the mixed polyketide-polypeptide structural class, were isolated from the marine cyanobacterium, Lyngbya majuscula, and were tested to determine if they showed activity against barnacle larval settlement. The assays revealed four compounds, dolastatin 16, hantupeptin C, majusculamide A, and isomalyngamide A, that showed moderate to potent anti-larval settlement activities, with EC(50) values ranging from 0.003 to 10.6 microg ml(-1). In addition, field testing conducted over a period of 28 days (using the modified Phytagel method) based on the cyanobacterial compound, dolastatin 16, showed significantly reduced barnacle settlement as compared to controls at all the concentrations tested. The results of this study highlight the importance of marine cyanobacteria as an underexplored source of potential environmentally friendly antifoulants.

Effects of human trampling on a rocky shore fauna on the Sao Paulo coast, southeastern Brazil / Impactos do pisoteio humano na fauna de um costão rochoso do litoral de São Paulo, no sudeste brasileiro

Increased tourist activity in coastal regions demands management strategies to reduce impacts on rocky shores. The highly populated coastal areas in southeastern Brazil are an example of degradation caused by development of industry and tourism. Among different shore impacts, trampling has been intensively studied, and may represent a significant source of stress for intertidal fauna. A randomised blocks design was applied to experimentally study the effects of two different trampling intensities on richness, diversity, density and biomass of the rocky shore fauna of Obuseiro beach, Guarujá, southeastern Brazil. Blocks were distributed in two portions of the intertidal zone, dominated respectively by Chthamalus bisinuatus (Cirripedia) and Isognomon bicolor (Bivalvia). Blocks were trampled over three months, simulating the vacation period in Brazil and were monitored for the following nine months. Results indicate that Chthamalus bisinuatus is vulnerable to trampling impacts. Richness, diversity and turn-over index tended to be higher in trampled plots four months after trampling ceased. In general, results agree with previous trampling studies, suggesting that even low intensities of trampling may cause some impact on intertidal communities. Management strategies should include isolation of sensitive areas, construction of boardwalks, visitor education and monitoring programmes. In Brazil, additional data obtained from experimental studies are necessary in order to achieve a better understanding of trampling impacts on rocky shore communities.

O aumento da atividade turística em áreas costeiras nas últimas décadas faz necessária a adoção de estratégias de manejo para reduzir os impactos gerados às comunidades de costões rochosos. A região costeira do sudeste brasileiro possui bons exemplos de degradação causada pelo turismo e desenvolvimento industrial. Dentre os diferentes distúrbios causados pela visitação, o pisoteio têm sido estudado de forma intensa e pode representar uma fonte significativa de impactos para as comunidades da zona entre-marés. Neste projeto, foi aplicado um desenho de blocos randômicos para se avaliar experimentalmente os efeitos de duas intensidades de pisoteio na riqueza, diversidade, densidade, recobrimento e biomassa da fauna de um costão situado na praia do Obuseiro, no município do Guarujá, São Paulo, Brasil. Os blocos foram alocados em dois povoamentos diferentes, dominados respectivamente por Chthamalus bisinuatus (Cirripedia) e Isognomon bicolor (Bivalvia). O pisoteio foi aplicado durante três meses, simulando a temporada de férias no Brasil, e os blocos foram monitorados nos nove meses seguintes. Os resultados indicaram que Chthamalus bisinuatus é vulnerável aos impactos do pisoteio. Os índices de riqueza, diversidade e turnover apresentaram uma tendência ao aumento nas áreas pisoteadas quatro meses após o término do pisoteio. No geral, os resultados concordam com estudos anteriores e sugerem que mesmo baixas intensidades do distúrbio podem causar impacto às comunidades bentônicas. Estratégias de manejo devem envolver o isolamento de áreas sensíveis, a construção de passarelas, a educação dos visitantes e o monitoramento das comunidades impactadas. No Brasil, a realização de maior quantidade de estudos experimentais é necessária para a melhor compreensão dos impactos do pisoteio nas comunidades de costões rochosos.

Biology and ecology of the hydrocoral millepora on coral reefs.

Millepores are colonial polypoidal hydrozoans secreting an internal calcareous skeleton of an encrusting or upright form, often of considerable size. Defensive polyps protruding from the skeleton are numerous and highly toxic and for this reason millepores are popularly known as "stinging corals" or "fire corals." In shallow tropical seas millepore colonies are conspicuous on coral reefs and may be locally abundant and important reef-framework builders. The history of systematic research on the Milleporidae and the sister family Stylasteridae is rich and full with the works of early naturalists beginning with Linnaeus. Seventeen living millepore species are recognised. Marked phenotypic variation in form and structure of colonies is characteristic of the genus Millepora. The first published descriptions of the anatomy and histology of millepores were by H. N. Moseley in one of the Challenger Expedition reports. These original, detailed accounts by Moseley remain valid and, except for recent descriptions of the ultrastructure of the skeleton and skeletogenic tissues, have not needed much modification. Millepores occur worldwide on coral reefs at depths of between 1 and 40 m and their distribution on reefs is generally zoned in response to physical factors. Colonies may be abundant locally on coral reefs but usually comprise <10% of the overall surface cover. Growth rates of colonies are similar to the measured rates of branching and platelike scleractinian corals. Millepores are voracious zooplankton feeders and they obtain part of their nutrition from autotrophic sources, photosynthetic production by symbiotic zooxanthellae. Reproduction in millepores is characterised by alternation of generations with a well-developed polypoid stage that buds off planktonic medusae. Sexual reproduction is seasonal for known species and the medusae have a brief planktonic life. Asexual production is achieved by sympodial growth, the production of new skeleton and soft tissue along a growing edge or branch tip, and by the reattachment, regeneration and repair of damaged or broken colony fragments. The physiological and ecological responses of species of millepores are similar to those of the species of scleractinian corals over a broad range of natural and anthropogenic disturbances. Severe damage to colonies may occur during major storms. Delicately branching species are more susceptible than massive and bladed species. The ability of broken fragments to regenerate can ameliorate the extent of damage. Widespread bleaching and mortality of millepores has been reported during mass bleaching events that have affected many coral reefs. Millepores are often the first to recover after short-term bleaching events. Harmful effects of oil spills, chronic oil pollution and oil-spill detergents have been widely reported for millepores. Although the hydrozoan coenosarc, with its fiercely stinging zooids, does not appear to be an attractive substratum for attachment and settlement of epizooans, a number of sessile and errant forms commonly occur on millepores. These include barnacles, amphipods, tanaid and alpheid crustaceans, polychaetes and gastropods. Burrowing molluscs, polychaetes and crustacea also abound. Many of these species or their close relatives also occur on scleractinian corals. A variety of predators, grazers and fouling organisms occur on millepores. These include errant polychaetes, several coral-feeding fish and a gastropod mollusc. Various invasive green, red and brown algae are widespread, growing on dead branches of millepores and overgrowing live coral tissue. Various "band diseases" associated with microorganisms that appear to cause lesions on millepores and loss of tissue have been documented but are not of widespread occurrence. Infestations of endolithic algae and fungi growing within the skeletons have been reported in a number of millepore species.

Variation among families for characteristics of the adhesive plaque in the barnacle Balanus amphitrite.

A quantitative genetics approach was used to examine variation in the characteristics of the adhesive plaque of the barnacle Balanus amphitrite Darwin attached to two silicone substrata. Barnacles settled on silicone polymer films occasionally form thick, soft adhesive plaques, in contrast to the thin, hard plaques characteristic of attachment to other surfaces. The proportion of barnacles producing a thick adhesive plaque was 0.31 for Veridian, a commercially available silicone fouling-release coating, and 0.18 for Silastic T-2, a silicone rubber used for mold-making. For both materials, significant variation among maternal families in the proportion of barnacles producing a thick adhesive plaque was observed, which suggests the presence of genetic variation, or maternal environmental effects, for this plaque characteristic. For the Veridian coating, barnacles expressing the thick adhesive plaque also exhibited significantly reduced tenacity. This represents the first reported case for potential genetic control of intraspecific phenotypic variation in the physical characteristics and tenacity of the adhesive of a fouling invertebrate.

Unraveling the evolutionary radiation of the thoracican barnacles using molecular and morphological evidence: a comparison of several divergence time estimation approaches.

The Thoracica includes the ordinary barnacles found along the sea shore and is the most diverse and well-studied superorder of Cirripedia. However, although the literature abounds with scenarios explaining the evolution of these barnacles, very few studies have attempted to test these hypotheses in a phylogenetic context. The few attempts at phylogenetic analyses have suffered from a lack of phylogenetic signal and small numbers of taxa. We collected DNA sequences from the nuclear 18S, 28S, and histone H3 genes and the mitochondrial 12S and 16S genes (4,871 bp total) and data for 37 adult and 53 larval morphological characters from 43 taxa representing all the extant thoracican suborders (except the monospecific Brachylepadomorpha). Four Rhizocephala (highly modified parasitic barnacles) taxa and a Rhizocephala + Acrothoracica (burrowing barnacles) hypothetical ancestor were used as the outgroup for the molecular and morphological analyses, respectively. We analyzed these data separately and combined using maximum likelihood (ML) under "hill-climbing" and genetic algorithm heuristic searches, maximum parsimony procedures, and Bayesian inference coupled with Markov chain Monte Carlo techniques under mixed and homogeneous models of nucleotide substitution. The resulting phylogenetic trees answered key questions in barnacle evolution. The four-plated Iblomorpha were shown as the most primitive thoracican, and the plateless Heteralepadomorpha were placed as the sister group of the Lepadomorpha. These relationships suggest for the first time in an invertebrate that exoskeleton biomineralization may have evolved from phosphatic to calcitic. Sessilia (nonpedunculate) barnacles were depicted as monophyletic and appear to have evolved from a stalked (pedunculate) multiplated (5+) scalpelloidlike ancestor rather than a five-plated lepadomorphan ancestor. The Balanomorpha (symmetric sessile barnacles) appear to have the following relationship: (Chthamaloidea(Coronuloidea(Tetraclitoidea, Balanoidea))). Thoracican divergence times were estimated under ML-based local clock, Bayesian, and penalized likelihood approaches using an 18S data set and three calibration points: Heteralepadomorpha = 530 million years ago (MYA), Scalpellomorpha = 340 MYA, and Verrucomorpha = 120 MYA. Estimated dates varied considerably within and between approaches depending on the calibration point. Highly parameterized local clock models that assume independent rates (r > or = 15) for confamilial or congeneric species generated the most congruent estimates among calibrations and agreed more closely with the barnacle fossil record. Reasonable estimates were also obtained under the Bayesian procedure of Kishino et al. (2001, Mol. Biol. Evol. 18:352-361) but using multiple calibrations. Most of the dates estimated under the Bayesian procedure of Aris-Brosou and Yang (2002, Syst. Biol. 51:703-714) and the penalized likelihood method using single and/or multiple calibrations were inconsistent among calibrations and did not fit the fossil record.