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Photoreceptor cells in the eyes of Bilateria are often classified into microvillar cells with rhabdomeric opsin and ciliary cells with ciliary opsin, each type having specialized molecular components and physiology. First data on the recently discovered xenopsin point towards a more complex situation in protostomes. In this study, we provide clear evidence that xenopsin enters cilia in the eye of the larval bryozoan Tricellaria inopinata and triggers phototaxis. As reported from a mollusc, we find xenopsin coexpressed with rhabdomeric-opsin in eye photoreceptor cells bearing both microvilli and cilia in larva of the annelid Malacoceros fuliginosus . This is the first organism known to have both xenopsin and ciliary opsin, showing that these opsins are not necessarily mutually exclusive. Compiling existing data, we propose that xenopsin may play an important role in many protostome eyes and provides new insights into the function, evolution, and possible plasticity of animal eye photoreceptor cells., Competing Interests: CD, SK, ST, IK, HH No competing interests declared, (© 2020, Döring et al.)

Bryozoans are sessile, filter-feeding, and colony-building invertebrate organisms. Fredericella sultana is a well known primary host of the myxozoan parasite Tetracapsuloides bryosalmonae . There have been no attempts to identify the cellular responses induced in F. sultana during the T. bryosalmonae development. We therefore performed transcriptome analysis with the aim of identifying candidate genes and biological pathways of F. sultana involved in the response to T. bryosalmonae . A total of 1166 differentially up- and downregulated genes were identified in the infected F. sultana . Gene ontology of biological processes of upregulated genes pointed to the involvement of the innate immune response, establishment of protein localization, and ribosome biogenesis, while the downregulated genes were involved in mitotic spindle assembly, viral entry into the host cell, and response to nitric oxide. Eukaryotic Initiation Factor 2 signaling was identified as a top canonical pathway and MYCN as a top upstream regulator in the differentially expressed genes. Our study provides the first transcriptional profiling data on the F. sultana zooid's response to T. bryosalmonae . Pathways and upstream regulators help us to understand the complex interplay in the infected F. sultana . The results will facilitate the elucidation of innate immune mechanisms of bryozoan and will lay a foundation for further analyses on bryozoan-responsive candidate genes, which will be an important resource for the comparative analysis of gene expression in bryozoans.

Less than one percent of marine natural products characterized since 1963 have been obtained from the phylum Bryozoa which, therefore, still represents a huge reservoir for the discovery of bioactive metabolites with its ~6000 described species. The current review is designed to highlight how bryozoans use sophisticated chemical defenses against their numerous predators and competitors, and which can be harbored for medicinal uses. This review collates all currently available chemoecological data about bryozoans and lists potential applications/benefits for human health. The core of the current review relates to the potential of bryozoan metabolites in human diseases with particular attention to viral, brain, and parasitic diseases. It additionally weighs the pros and cons of total syntheses of some bryozoan metabolites versus the synthesis of non-natural analogues, and explores the hopes put into the development of biotechnological approaches to provide sustainable amounts of bryozoan metabolites without harming the natural environment.

The breadth of habitat occupied by a species, and the rate at which a species can expand into new habitats has important ecological and evolutionary consequences. Here we explore when extant species of free-living cupuladriid bryozoans expanded into new benthic Caribbean habitats that emerged during the final stages of formation of the Isthmus of Panama. Habitat breadth was estimated using the abundances of over 90,000 colonies in ten cupuladriid species, along with the ecological and sedimentary characteristics of the samples in which they occurred. Data reveal that all species expanded their habitat breadths during the last 6 Myr, but did so at a different tempo. 'Young' species - those that originated after 5 Ma - expanded relatively quickly, whereas 'old' species - those that originated before 9 Ma - took a further 2 Myr to achieve a comparable level of expansion. We propose that, like invasive species, young species are less restrained when expanding their habitat breadths compared to older well-established species. Understanding the mechanism causing this restraint requires further research.

Marine bryozoans play an important role for the discovery of novel bioactive compounds among marine organisms. In this review, we summarize 164 new secondary metabolites including macrocyclic lactones, sterols, alkaloids, sphingolipids and so forth from 24 marine bryozoans in the last two decades. The structural features, bioactivity, structure-activity relationship, mechanism and strategies to address the resupply of these scarce secondary metabolites are discussed. The structural and bioactive diversity of the secondary metabolites from marine bryozoans indicated the possibility of using these compounds, especially bryostatin 1 (1), bryostatin analog (BA1), alkaloids (50, 53, 127-128 and 134-139), sphingolipids sulfates (148 and 149) and sulfur-containing aromatic compound (160), as the starting points for new drug discovery.

The bryozoan Bugula neritina is a cosmopolitan marine fouling species that causes major fouling problems in sub-tropical waters. Settlement of B. neritina larvae can be triggered without an obvious external cue. Here, the negative regulatory role of nitric oxide (NO) during larval settlement of B. neritina was demonstrated to be mediated by cyclic guanosine monophosphate (cGMP). Although the regulatory role of the NO-p38 MAPK signaling axis in larval settlement was not evident, inhibition of nitric oxide synthase (NOS) led to the deactivation of p38 MAPK. Exclusive localization of NO and NO signaling components in sensory-related organs of the larvae is consistent with its signal transduction function in metamorphosis. Overall, this study provides new insights into the regulatory roles of the NO-p38MAPK/cGMP pathway in B. neritina settlement.

Covering: up to 2018 Symbiotic microbes interact with animals, often by producing natural products (specialized metabolites; secondary metabolites) that exert a biological role. A major goal is to determine which microbes produce biologically important compounds, a deceptively challenging task that often rests on correlative results, rather than hypothesis testing. Here, we examine the challenges and successes from the perspective of marine animal-bacterial mutualisms. These animals have historically provided a useful model because of their technical accessibility. By comparing biological systems, we suggest a common framework for establishing chemical interactions between animals and microbes.

β-Branching is an expansion upon canonical polyketide synthase extension that allows for the installation of diverse chemical moieties in several natural products. Several of these moieties are unique among natural products, including the two vinyl methylesters found in the core structure of bryostatins. This family of molecules is derived from an obligate bacterial symbiont of a sessile marine bryozoan, Bugula neritina. Within this family, bryostatin 1 has been investigated as an anticancer, neuroprotective, and immunomodulatory compound. We have turned to the biosynthetic gene cluster within the bacterial symbiont to investigate the biosynthesis of bryostatins. Recent sequencing efforts resulted in the annotation of two missing genes: bryT and bryU. Using novel chemoenzymatic techniques, we have validated these as the missing enoyl-CoA hydratase and donor acyl carrier protein, essential components of the β-branching cassette of the bryostatin pathway. Together, this cassette installs the vinyl methylester moieties essential to the activity of bryostatins., (© 2018 Elsevier Inc. All rights reserved.)

Marine invertebrates are a promising source of novel natural products with biological activities. The phylum Bryozoa is relatively under-investigated in this context, although a number of compounds with medical potential has been discovered in recent years. Here, we report on the novel group of brominated metabolites from the bryozoan Terminoflustra membranaceatruncata, including analysis of biological activities of the tribrominated terminoflustrindole A (Cm-1) and the structures of the related dibrominated variants terminoflustrindoles B and C. Terminoflustrindole A was previously shown to have fungicidal properties. Although they vary by just one bromine group in each case from terminoflustrindole A, in this study, we report that terminoflustrindoles B and C exhibit no antimicrobial activity in the same assays. In addition to displaying antifungal activity, Terminoflustrindole A was also found to exhibit potent cytotoxic activity when tested against tumour cell lines. The gradient distribution of this compound within the bryozoan colony was demonstrated using LC-MS-analysis.

A new sterol, (23 R )-methoxycholest-5,24-dien-3β-ol ( 1 ), two new ceramides, (2 S ,3 R ,4 E ,8 E )-2-(tetradecanoylamino)-4,8-octadecadien-l,3-diol ( 6 ) and (2 S ,3 R ,2' R ,4 E ,8 E )-2-(tetradecanoylamino)-4,8-octadecadien-l,3,2'-triol ( 7 ), together with three known sterols ( 2 - 4 ), a lactone ( 5 ) and two ceramides ( 8 , 9 ), were isolated from the marine bryozoan Cryptosula pallasiana , collected at Huang Island of China. The structures of the new compounds were elucidated by extensive spectroscopic analyses, chemical methods and quantum electronic circular dichroism (ECD) calculations. Among the isolated compounds, sterol 1 possessed a rare side chain with a methoxy group at C-23, and a double bond between C-24 and C-25. Ceramides 6 and 7 possessed 14 carbons in their long-chain fatty acid base (FAB), which were different from the normal ceramides with 16 carbons in the FAB. Moreover, compounds 5 and 8 were isolated for the first time from marine bryozoans. Compounds 1 - 9 were evaluated for their cytotoxicity against human tumor cell lines HL-60, Hep-G2 and SGC-7901. The results showed that lactone 5 appears to have strong cytotoxicity against the test tumor cell lines, with IC 50 values from 4.12 μM to 7.32 μM, and sterol 1 displayed moderate cytotoxicity with IC 50 values between 12.34 μM and 18.37 μM, while ceramides 6 - 9 showed weak cytotoxicity with IC 50 ranging from 21.13 μM to 58.15 μM.

Accessing many fundamental questions in biology begins with empirical estimation of simple monotonic rates of underlying biological processes. Across a variety of disciplines, ranging from physiology to biogeochemistry, these rates are routinely estimated from non-linear and noisy time series data using linear regression and ad hoc manual truncation of non-linearities. Here, we introduce the R package LoLinR, a flexible toolkit to implement local linear regression techniques to objectively and reproducibly estimate monotonic biological rates from non-linear time series data, and demonstrate possible applications using metabolic rate data. LoLinR provides methods to easily and reliably estimate monotonic rates from time series data in a way that is statistically robust, facilitates reproducible research and is applicable to a wide variety of research disciplines in the biological sciences., (© 2017. Published by The Company of Biologists Ltd.)

In the White Sea bryozoans Arctonula arctica, the structure of the nervous system and distribution of 5-hydroxytryptamine (5-HT) and FMRF-amide were studied for the first time using immunohistochemical methods and confocal scanning microscopy. The neurotransmitters studied have been actively involved into the integrative processes, gut functioning, and regulation of motion activity. In avicularia, 5-HT and FMRF-amide receptors are capable of performing the same functions, except for participation in the gut functioning, because they have no digestive system.

Plastics are common and pervasive anthropogenic debris in marine environments. Floating plastics provide opportunities to alter the abundance, distribution and invasion potential of sessile organisms that colonize them. We selected plastics from seven recycle categories and quantified settlement of (i) bryozoans Bugula neritina (Linnaeus, 1758) in the lab and in the field, and of (ii) barnacles Amphibalanus (= Balanus) amphitrite (Darwin, 1854) in the field. In the laboratory we cultured barnacles on the plastics for 8 weeks and quantified growth, mortality, and breaking strength of the side plates. In the field all recyclable plastics were settlement substrata for bryozoans and barnacles. Settlement depended on the type of plastic. Fewer barnacles settled on plastic surfaces compared to glass. In the lab and in the field, bryozoan settlement was higher on plastics than on glass. In static laboratory rearing, barnacles growing on plastics were initially significantly smaller than on glass. This suggested juvenile barnacles were adversely impacted by materials leaching from the plastics. Barnacle mortality was not significantly different between plastic and glass surfaces, but breaking strength of side plates of barnacles on polyvinyl chloride (PVC) and polycarbonate (PC) were significantly lower than breakage strength on glass. Plastics impact marine ecosystems directly by providing new surfaces for colonization with fouling organisms and by contaminants shown previously to leach out of plastics and impact biological processes., (Published by Elsevier Ltd.)

Background: Assessing pathway activity levels is a plausible way to quantify metabolic differences between various conditions. This is usually inferred from microarray expression data. Wide availability of NGS technology has triggered a demand for bioinformatics tools capable of analyzing pathway activity directly from RNA-Seq data. In this paper we introduce XPathway, a set of tools that compares pathway activity analyzing mapping of contigs assembled from RNA-Seq reads to KEGG pathways. The XPathway analysis of pathway activity is based on expectation maximization and topological properties of pathway graphs., Results: XPathway tools have been applied to RNA-Seq data from the marine bryozoan Bugula neritina with and without its symbiotic bacterium "Candidatus Endobugula sertula". We successfully identified several metabolic pathways with differential activity levels. The expression of enzymes from the identified pathways has been further validated through quantitative PCR (qPCR)., Conclusions: Our results show that XPathway is able to detect and quantify the metabolic difference in two samples. The software is implemented in C, Python and shell scripting and is capable of running on Linux/Unix platforms. The source code and installation instructions are available at http://alan.cs.gsu.edu/NGS/?q=content/xpathway .

In marine systems, oxygen availability varies at small temporal and spatial scales, such that current oxygen levels may not reflect conditions of the past. Different studies have shown that marine invertebrate larvae can select settlement sites based on local oxygen levels and oxygenation history of the biofilm, but no study has examined the interaction of both. The influence of normoxic and hypoxic water and oxygenation history of biofilms on pre-settlement behavior and settlement of the bryozoan Bugula neritina was tested. Larvae used cues in a hierarchical way: the oxygen levels in the water prime larvae to respond, the response to different biofilms is contingent on oxygen levels in the water. When oxygen levels varied throughout biofilm formation, larvae responded differently depending on the history of the biofilm. It appears that B. neritina larvae integrate cues about current and historical oxygen levels to select the appropriate microhabitat and maximize their fitness.

Bryozoans are typical modular organisms. They consist of repetitive structural units, the zooids. Bryozoan colonies grow by zooidal budding, with the distribution pattern of the budding loci underlying the diversity of colony forms. Budding is usually restricted to the zooids at the periphery of the colony, which form a "growing edge" or local terminal growth zones. Non-budding parts of the colony can be functionally subdivided, too. In many species colonies consists of regular, often repetitive zones of feeding and non-feeding modules, associated with a periodical degeneration and regeneration of the polypide, retractile tentacle crown with a gut and the accompanying musculature. So, there is functional differentiation in bryozoan colonies but its mechanisms are unknown. Presumably, budding and/or polypide recycling in different colony parts are induced or inhibited by certain determinants of functional specialization. An effective tool of their identification is the comparison of proteomes of functionally different zones. Here we report the results of proteomic analysis of three bryozoan species from the White Sea, which have a different colony form: Flustrellidra hispida, Terminoflustra membranaceotruncata and Securiflustra securifrons. Using differential two-dimensional electrophoresis (2D-DIGE), we compared proteomes of the growing edge and the zones consisting of feeding and non-feeding zooids in these species. We estimated the overall proteome variability, revealed proteins whose relative abundance gradually changed along the proximal-distal colony axis and suggested that they might be involved in the functional differentiation of the colony.

Chitin immediately suggests the representatives of the kingdom Fungi, as well as such phyla as Annelida, Mollusca, Porifera, Cnidaria and, mostly, Arthropoda. Although Bryozoa also represents a chitin-containing phylum, no study has been developed yet on the isolation or characterization of the chitin from it. In this study, physiochemical properties of the chitin isolated from Plumatella repens belonging to the phylum Bryozoa was determined for the first time. The chitin structure was also studied comparatively by isolating chitin from an insect species (Palomena prasina) of the phylum Arthropoda, and Fomes fomentarius belonging to the kingdom Fungi. It was observed that the bryozoan chitin was in the α form, as in the arthropod and fungal chitins. The chitin contents in the dry weight of the bryozoan, fungal and insect species were observed to be 13.3%, 2.4%, and 10.8%, respectively. The insect chitin exhibited the highest thermal stability followed by that of the bryozoan and then the fungal chitins. Surface morphologies reveal that the insect and bryozoan chitins were composed of nano fibre and pore structures, whereas the fungal chitin had no pores or fibres. The crystallinity of the insect chitin (CrI=84.9%) was higher than the bryozoan (CrI=60.1%) and fungal chitins (CrI=58.5%)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The antimicrobial activity of Antarctic bryozoans and the ecological functions of the chemical compounds involved remain largely unknown. To determine the significant ecological and applied antimicrobial effects, 16 ether and 16 butanol extracts obtained from 13 different bryozoan species were tested against six Antarctic (including Psychrobacter luti, Shewanella livingstonensis and 4 new isolated strains) and two bacterial strains from culture collections (Escherichia coli and Bacillus cereus). Results from the bioassays reveal that all ether extracts exhibited antimicrobial activity against some bacteria. Only one butanol extract produced inhibition, indicating that antimicrobial compounds are mainly lipophilic. Ether extracts of the genus Camptoplites inhibited the majority of bacterial strains, thus indicating a broad-spectrum of antimicrobial activity. Moreover, most ether extracts presented activities against bacterial strains from culture collections, suggesting the potential use of these extracts as antimicrobial drugs against pathogenic bacteria., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Five new ceramides, neritinaceramides A (1), B (2), C (3), D (4) and E (5), together with six known ceramides (6-11), two known alkyl glycerylethers (12 and 13) and a known nucleoside (14), were isolated from marine bryozoan Bugula neritina, which inhabits the South China Sea. The structures of the new compounds were elucidated as (2S,3R,3'S,4E,8E,10E)-2-(hexadecanoylamino)-4,8,10-octadecatriene-l,3,3'-triol (1), (2S,3R,2'R,4E,8E,10E)-2-(hexadecanoylamino)-4,8,10-octadecatriene-l,3,2'-triol (2), (2S,3R,2'R,4E,8E,10E)-2-(octadecanoylamino)-4,8,10-octadecatriene-l,3,2'-triol (3), (2S,3R,3'S,4E,8E)-2-(hexadecanoylamino)-4,8-octadecadiene-l,3,3'-triol (4) and (2S,3R,3'S,4E)-2-(hexadecanoylamino)-4-octadecene-l,3,3'-triol (5) on the basis of extensive spectral analysis and chemical evidences. The characteristic C-3'S hydroxyl group in the fatty acid moiety in compounds 1, 4 and 5, was a novel structural feature of ceramides. The rare 4E,8E,10E-triene structure in the sphingoid base of compounds 1-3, was found from marine bryozoans for the first time. The new ceramides 1-5 were evaluated for their cytotoxicity against HepG2, NCI-H460 and SGC7901 tumor cell lines, and all of them exhibited selective cytotoxicity against HepG2 and SGC7901 cells with a range of IC50 values from 47.3 μM to 58.1 μM. These chemical and cytotoxic studies on the new neritinaceramides A-E (1-5) added to the chemical diversity of B. neritina and expanded our knowledge of the chemical modifications and biological activity of ceramides.

Mutualism, a beneficial relationship between two species, often requires intimate interaction between the host and symbiont to establish and maintain the partnership. The colonial marine bryozoan Bugula neritina harbors an as yet uncultured endosymbiont, "Candidatus Endobugula sertula," throughout its life stages. The bacterial symbiont is the putative source of bioactive complex polyketide metabolites, the bryostatins, which chemically defend B. neritina larvae from predation. Despite the presence of "Ca. Endobugula sertula" in all life stages of the host, deterrent bryostatins appear to be concentrated in reproductive portions of the host colony, suggesting an interaction between the two partners to coordinate production and distribution of the metabolites within the colony. In this study, we identified host genes that were differentially expressed in control colonies and in colonies cured of the symbiont. Genes that code for products similar to glycosyl hydrolase family 9 and family 20 proteins, actin, and a Rho-GDP dissociation inhibitor were significantly downregulated (more than twice) in antibiotic-cured non-reproductive zooids compared to control symbiotic ones. Differential expression of these genes leads us to hypothesize that the host B. neritina may regulate the distribution of the symbiont within the colony via mechanisms of biofilm degradation and actin rearrangement, and consequently, influences bryostatin localization to bestow symbiont-associated protection to larvae developing in the reproductive zooids.

Biomonitors are commonly used to assess levels of bioavailable contaminants in the environment, however the relationships between biomonitor tissue concentrations and ecological effects are rarely assessed. The present study investigated metal contamination within a highly industrialised harbour and ecological effects on sessile invertebrates. The native oyster Saccostrea glomerata was deployed as a biomonitor across twenty-six sites to test for correlations between metal levels in their tissues and the recruitment of hard-substrate invertebrates. Concentrations of lead and copper in oyster tissues were negatively correlated with densities of the dominant barnacle, Amphibalanus variegatus, and positively correlated with densities of the dominant polychaete, Hydroides elegans, and the two native encrusting bryozoans Celloporaria nodulosa and Arachnopusia unicornis. Results suggest that highly localised events drive contaminant availability and that these events pose a significant risk to fauna. Biomonitoring studies may be enhanced by running concurrent ecological surveys., (This journal is © The Royal Society of Chemistry 2012)

Blooms of the toxic alga Karenia brevis occur along coastlines where sessile suspension feeding invertebrates are common components of benthic communities. We studied the effects of K. brevis on four benthic suspension feeding invertebrates common to the coast of the SE United States: the sponge Haliclona tubifera, the bryozoan Bugula neritina, the bivalve Mercenaria mercenaria, and the tunicate Styela plicata. In controlled laboratory experiments, we determined the rate at which K. brevis was cleared from the seawater by these invertebrates, the effect of K. brevis on clearance rates of a non-toxic phytoplankton species, Rhodomonas sp., and the extent to which brevetoxins bioaccumulated in tissues of invertebrates using an enzyme-linked immunosorbent assay (ELISA). All four invertebrate species cleared significant quantities of K. brevis from seawater, with mean clearance rates ranging from 2.27 to 6.71 L g h⁻¹ for H. tubifera and S. plicata, respectively. In the presence of K. brevis, clearance rates of Rhodomonas sp. by B. neritina and S. plicata were depressed by 75% and 69%, respectively, while clearance rates by H. tubifera and M. mercenaria were unaffected. Negative effects of K. brevis were impermanent; after a recovery period of 13 h, B. neritina and S. plicata regained normal clearance rates. All four invertebrates accumulated high concentrations of brevetoxin after a 4h exposure to K. brevis, but when animals were transferred to filtered seawater for 15 h after exposure, brevetoxin concentrations in the tissues of H. tubifera and B. neritina decreased by ∼80%, while there was no change in toxin concentration in the tissues of S. plicata and M. mercenaria. High cell concentrations of K. brevis may cause a suppression of clearance rates in benthic suspension feeding invertebrates, resulting in a positive feedback for bloom formation. Also, high concentrations of toxin may accumulate in the tissues of benthic suspension feeding invertebrates that may be transferred to higher-level consumers., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Isocyanide is a potential antifouling compound in marine environments. In this study, we investigated its mode of action in three aquatic organisms. Two of them, the bryozoan Bugula neritina and the barnacle Balanus amphitrite, are major marine fouling invertebrates, and the other organism is the non-target species zebrafish Danio rerio. In the swimming larvae of B. neritina, isocyanide did not affect the total attachment rate (≤50 µg ml(-1)), but it did change the attachment site by increasing the percentage of attachment on the bottom of the container rather than on the wall or air-water inter-surface. Isocyanide binds several proteins in B. neritina as identified via SDS-PAGE-LC-MS/MS: 1) a 30 kD protein band containing two proteins similar to voltage dependent anion channels (VDAC), which control the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol and the release of apoptogenic factors from mitochondria of mammalian cells; and 2) an unknown 39 kD protein. In B. amphitrite cyprids, the isocyanide binding protein were 1) a protein similar to NADH-ubiquinone oxidoreductase, which is the "entry enzyme" of oxidative phosphorylation in mitochondria; and 2) cytochrome P450. In Danio rerio embryos, isocyanide caused "wavy" notochords, hydrocephalus, pericardial edema, poor blood circulation, and defects in pigmentation and hematopoiesis, which phenocopied copper deficiency. This is the first report on isocyanide binding proteins in fouling organisms, as well as the first description of its phenotype and potential toxicology in zebrafish.

In this study, we analyzed the metamorphosis of the marine bryozoan Bugula neritina. We observed the morphogenesis of the ancestrula. We defined three distinct pre-ancestrula stages based on the anatomy of the developing polypide and the overall morphology of pre-ancestrula. We then used an annotation based enrichment analysis tool to analyze the B. neritina transcriptome and identified over-representation of genes related to Wnt signaling pathways, suggesting its involvement in metamorphosis. Finally, we studied the temporal-spatial gene expression studies of several Wnt pathway genes. We found that one of the Wnt ligand, BnWnt10, was expressed spatially opposite to the Wnt antagonist BnsFRP within the blastemas, which is the presumptive polypide. Down-stream components of the canonical Wnt signaling pathway were exclusively expressed in the blastemas. Bnβcatenin and BnFz5/8 were exclusively expressed in the blastemas throughout the metamorphosis. Based on the genes expression patterns, we propose that BnWnt10 and BnsFRP may relate to the patterning of the polypide, in which the two genes served as positional signals and contributed to the polarization of the blastemas. Another Wnt ligand, BnWnt6, was expressed in the apical part of the pre-ancestrula epidermis. Overall, our findings suggest that the Wnt signaling pathway may be important to the pattern formation of polypide and the development of epidermis.

Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid-phase IEF sample prefractionation combined with 2-DE and MALDI-TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium-binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required.

Larvae of some benthic invertebrates respond (metamorphose or not) to chemical cues within minutes or hours and often without excessive transcription or translation. Although protein phosphorylation is one of the most important molecular switching mechanisms that govern variety of rapid cellular responses in higher organisms, this is the first study to analyze the global protein expression and protein phosphorylation status during larval metamorphosis in two major marine biofouling invertebrates (a bryozoan Bugula neritina and a barnacle Balanus amphitrite). Results indicate that larval proteomic response to metamorphosis (inhibiton or induction) involves substantial change in the phosphorylation status of proteins rather than de novo protein synthesis. An abundantly expressed and an unnamed phosphoprotein that appears to play key regulatory role in larval metamorphosis was identified. When larvae of bryozoan and barnacle were challenged with a metamorphosis (and kinase) inhibitor, the genistein, the number of phosphoproteins in bryozoan were substantially reduced but drastically increased in barnacle. Taken together, this is the first time that the usefulness of employing 2DE-based proteomic and phosphoproteomic approaches was demonstrated for us to understand the molecular mechanisms of larval metamorphosis and to study the mode-of-action of chemical cues in marine organisms.

The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete open reading frame (ORF) (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.

'Candidatus Endobugula sertula,' the uncultivated gamma-proteobacterial symbiont of the marine bryozoan Bugula neritina, synthesizes bryostatins, complex polyketides that render B. neritina larvae unpalatable to predators. Although the symbiosis is well described, little is known about the locations of 'E. sertula' or the bryostatins throughout larval settlement, metamorphosis and early development. In this study, we simultaneously localized 'E. sertula' and the bryostatins in multiple stages of the B. neritina life cycle, using a novel bryostatin detection method based on its known ability to bind mammalian protein kinase C. Our results suggest that the bryostatins are deposited onto the exterior of B. neritina larvae during embryonic development, persist on the larval surface throughout metamorphosis and are shed prior to cuticle formation. During metamorphosis, 'E. sertula' remains adhered to the larval pallial epithelium and is incorporated into the preancestrula cystid tissue layer, which ultimately develops into a bud and gives rise to the next zooid in the colony. Colocalization of bryostatin signal with aggregates of 'E. sertula' in buds of ancestrulae suggested new synthesis of bryostatins in ancestrulae. In adult B. neritina colonies, symbiont microcolonies were observed in the funicular cords of rhizoids, which likely result in asexual transmission of 'E. sertula' to regenerated colonies. Furthermore, bryostatin signal was detected on the surface of the rhizoids of adult B. neritina colonies. Through simultaneous localization of the bryostatins and the 'E. sertula,' we determined how 'E. sertula' is transmitted, and identified shifts in bryostatin localization throughout the life cycle of the host B. neritina.

This Highlight covers the chemical ecology of bryozoans, primarily the ecological functions of bryozoan natural products. The Highlight is arranged taxonomically, according to the bryozoan Treatise classification (P. Bock, Bryozoa Homepage, 2006, http://bryozoa.net).

Monthly variations of total glutathione, glutathione-S-transferases, glutathione peroxidase, glutathione reductase, glyoxalase I, glyoxalase II, and catalase were studied in Lophopus crystallinus, a freshwater bryozoan, collected from Lake Piediluco. Specimens were sampled during the vegetative period from November to April for two different cycles: cycle I from 1996 to 1997 and cycle II from 1999 to 2000. Simultaneously, analyses of heavy metal content such as lead, cadmium, chromium, nickel, copper, iron, and zinc were determined in the samples of L. crystallinus. Specimens of cycle II displayed highest chromium and iron accumulation and elevated levels of biochemical parameters. Total glutathione content, catalase, and glutathione peroxidase activities, were related with the highest metal concentrations in L. crystallinus from Lake Piediluco.

Marine Natural Products (MNPs), such as bryostatin 1, are exposed to a range of physical and chemical conditions through the life cycle of the host organism. These include exposure to sunlight, oxidizing and reducing agents, cation binding, and adsorption to reactive metal oxide surfaces. Using Fourier Transform-Ion Cyclotron Resonance (FT-ICR), Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS), UV/Vis absorbance spectroscopy, and molecular modeling, we studied the impact of UV light, TiO2, I2, and reaction with FeCl3 on the structure of bryostatin 1. Our results demonstrate that natural conditions transform bryostatin to a number of structures, including one with a molar mass of 806 Da, which we have previously identified in the sediment collected from the Gulf of Mexico. To date, at least 20 different structures of bryostatin have been reported in the literature. This work suggests that these variations may be products of the chemical environment in which the bryozoa Bugula neritina resides and are not the result of genetic variations within Bugula.

The laboratory culture of the barnacle, Balanus amphitrite has made it possible to supply cypris larvae for antifouling assays all year round. The settlement of cyprids obtained from cultured B. amphitrite was indistinguishable from cyprids reared from field-collected barnacles. In laboratory cyprid settlement assays of extracts from marine sessile organisms, antifouling activity was expressed as the 99% inhibitory concentration (IC99), and toxicity as the 30% lethal concentration (LC30). The lipophilic extract of the marine bryozoan, Zoobotryon pellucidum, which showed promising antifouling activity, yielded 2,5,6-tribromo-1-methylgramine (TBG) by bioassay-guided isolation. The inhibitory activity of TBG was 6 times as strong as that of bis-(n-butyltin)oxide (TBTO), while its toxicity to cypris larvae was one-tenth that of TBTO. A structure-activity relationship study with 155 indole derivatives led to the discovery of the non-toxic antifoulant candidates 5,6-dichlorogramine, 5-chloro-2-methylgramine, and 5,6-dichrolo-1-methylgramine (DCMG), the latter being selected as the antifouling paint ingredient for performance evaluation tests (panel tests) following the results of a preliminary safety tests. A silicone-based antifouling paint containing 5-10% of DCMG was prepared and tested in the field; the painted surfaces remained almost barnacle-free for 1.5 years similar to silicone coatings such as Biox. Since the leaching rate of DCMG from the paint surface could be controlled by the addition of an acrylic acid-styrene copolymer (ASP), the life of the antifouling performance is expected to be improved. Thus, an extremely non-toxic silicone-based antifouling paint containing DCMG is under development.

Dogger Bank itch is an allergic contact dermatitis to the (2-hydroxyethyl) dimethylsulphoxonium ion, a metabolite produced by the marine Bryozoan Alcyonidium diaphanum. The condition may become disabling in affected individuals, who are chiefly fishermen and dock labourers. It involves regions of skin directly exposed to sea water and areas where water may course. As A. diaphanum is common in the coastal waters of Britain, Ireland and neighbouring mainland Europe, it is important for dermatologists to be aware of Dogger Bank itch. Data published in 1966 suggested that 7% of trawler-men at the UK port of Lowestoft had the condition. The current epidemiology is unknown, but the disease still occurs despite shrinkage of the fishing industry, and the condition is not confined to North Sea trawler-men as had been thought previously. It has been reported in trawler-men from Le Havre and shell fishermen from Cornwall, and we report it here in a fisherman using fixed nets in the eastern English Channel.

The effects of various Flustra foliacea metabolites on different types of human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated. Whereas most of the compounds tested had a small blocking effect, one of them, deformylflustrabromine, selectively increased the current obtained in alpha4beta2 receptors when co-applied with acetylcholine (ACh). The current increase was reversible and concentration-dependent. This potentiating effect was still present at saturating concentrations of acetylcholine, and no changes in single-channel conductance or reversal potential were observed, thus suggesting a modification in the gating of alpha4beta2 receptors. Dwell time analysis of single channel records indicates that the mechanism of action of deformylflustrabromine could be both an increase of the opening rate constant and a decrease of the closing rate constant on alpha4beta2 receptors. Thus, deformylflustrabromine may constitute an excellent starting point for the future development of related agents able to potentiate human neuronal nicotinic receptor function.

The marine bryozoan metabolites caulibugulone A-E were prepared from a readily available isoquinoline dione. These natural products were found to be potent and selective inhibitors of the dual specificity phosphatase Cdc25B.

Large-scale, renewable supplies of chemical constituents derived from marine invertebrates have limited development of potential new natural product drugs. This paper describes the development of two in-sea aquaculture systems designed and engineered for production of large quantities of biomass for two species of marine invertebrates desired for their natural product chemical constituents. The two invertebrates and their products were: (1) the cosmopolitan, arborescent bryozoan Bugula neritina (Phylum Bryozoa) for its anticancer chemical constituent bryostatin 1; and (2) Ecteinascidia turbinate (Phylum Tunicata) the source of anticancer ecteinascidin 743. For the third invertebrate Phylum Porifera, and its representative sponge Acanthella cavernosa (desired for its anti-parasitic and anti-infective kalihinols) in-sea systems were not developed in favor of controlled environment tank aquaculture systems. For the bryozoan and tunicate, projected economics for commercial-scale in-sea production proved cost effective. This was in contrast to the controlled environment sponge culture tank system, which did not prove to be economical due to inherent slow growth and low natural product yields of the sponge in culture. A non-destructive method for "milking" natural product chemicals from sponges was tested and is described.

The North Sea bryozoan Flustra foliacea was investigated to determine its secondary metabolite content. Gas chromatography-mass spectrometry analysis of a dichloromethane extract of the bryozoan enabled 11 compounds to be identified. Preparative high-performance liquid chromatography of the extract resulted in the isolation of 10 brominated alkaloids (compounds 1 to 10) and one diterpene (compound 11). All of these compounds were tested to determine their activities in agar diffusion assays against bacteria derived from marine and terrestrial environments. Compounds 1, 3 to 7, 10, and 11 exhibited significant activities against one or more marine bacterial strains originally isolated from F. foliacea but only weak activities against all of the terrestrial bacteria. By using the biosensors Pseudomonas putida(pKR-C12), P. putida(pAS-C8), and Escherichia coli(pSB403) the antagonistic effect on N-acyl-homoserine lactone-dependent quorum-sensing systems was investigated. Compounds 8 and 10 caused reductions in the signal intensities in these bioassays ranging from 50 to 20% at a concentration of 20 micro g/ml.

The oceans are the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds (especially the tunicate metabolite ET-743) show pronounced pharmacological activities and are interesting candidates for new drugs primarily in the area of cancer treatment. Other compounds are currently being developed as an analgesic (ziconotide from the mollusc Conus magus) or to treat inflammation. Numerous natural products from marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (bacteria, microalgae) are at least involved in their biosynthesis or are in fact the true sources of these respective metabolites. This assumption is corroborated by several studies on natural products from sponges that proved these compounds to be localized in symbiotic bacteria or cyanobacteria. Recently, molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the bryozoan Bugula neritina.

Bryostatins are a class of antineoplastic compounds isolated from the bryozoans Bugula neritina. A wide range of scientific research is currently underway, studying different aspects of the bryostatins. In this review we try to summarize the latest findings, including all the topics involved, from marine biology to medicinal chemistry.

Sixteen species (44 samples) of marine polychaetes and 10 species (14 samples) of bryozoans from eastern Australia were analyzed by GC/MS for the key seafood flavor components 2- and 4-bromophenol, 2, 4- and 2,6-dibromophenol, and 2,4,6-tribromophenol. All five bromophenols were found in 91% of polychaetes and 64% of bryozoans. The remaining samples all contained at least three bromophenols. 2,4, 6-Tribromophenol was found in all polychaetes and bryozoans and, with few exceptions, was present in the highest concentrations. The total bromophenol content determined on a wet-weight basis varied widely between species: for polychaetes, from 58 ng/g for Australonuphis teres to 8.3 million ng/g for Barantolla lepte, and for bryozoans, from 36 ng/g for Cladostephus spongiosus to 1668 ng/g for Amathia wilsoni. Species of polychaetes with the highest concentrations of bromophenols were all collected from muddy environments. The possible effects that dietary polychaetes and bryozoans have on the ocean-, brine-, or iodine-like flavors of certain seafoods are discussed.

Secondary metabolites from marine bryozoans are reviewed. Two ctenosome bryozoans are dealt with, one, Alcyonidium gelatinosum containing a sulfoxonium ion acting as hapten in an allergic contact dermatitis and the other, Zoobotryon verticillatum yielding bromogramines. Five cheilostome bryozoans have given rise to the isolation of unique secondary natural products. Bugula neritina is the source of the antineoplastic bryostatins and Bugula purple while Flustra foliacea have yielded an array of bromoindole alkaloids and a brominated quinoline. Chartella papyracea also have bromoindole alkaloids while Sessibugula translucens have ecological active bipyrroles. A biological active xanthine derivative has been reported from Phidolopora pacifica. The structure and chemistry of these compounds are discussed as are their origin, function and biological activity.