Gene co-option, duplication and divergence of cement proteins underpin the evolution of bioadhesives across barnacle life histories.

Acquisition of new genes often results in the emergence of novel functions and is a key step in lineage-specific adaptation. As a group of sessile crustaceans, barnacles establish permanent attachment through initial cement secretion at the larval phase followed by continuous cement secretion in juveniles and adults. However, the origins and evolution of barnacle larval and adult cement proteins remain poorly understood. By performing microdissection of larval cement glands, transcriptome and shotgun proteomics and immunohistochemistry validation, we identified 30 larval and 27 adult cement proteins of the epibiotic turtle barnacle Chelonibia testudinaria, of which the majority are stage- and barnacle-specific. While only two proteins, SIPC and CP100K, were expressed in both larvae and adults, detection of protease inhibitors and the cross-linking enzyme lysyl oxidase paralogs in larvae and adult cement. Other barnacle-specific cement proteins such as CP100k and CP52k likely share a common origin dating back at least to the divergence of Rhizocephala and Thoracica. Different CP52k paralogues could be detected in larval and adult cement, suggesting stage-specific cement proteins may arise from duplication followed by changes in expression timing of the duplicates. Interestingly, the biochemical properties of larval- and adult-specific CP52k paralogues exhibited remarkable differences. We conclude that barnacle larval and adult cement systems evolved independently, and both emerged from co-option of existing genes and de novo formation, duplication and functional divergence of lineage-specific cement protein genes. Our findings provide important insights into the evolutionary mechanisms of bioadhesives in sessile marine invertebrates.

Histology and transcriptomic analyses of barnacles with different base materials and habitats shed lights on the duplication and chemical diversification of barnacle cement proteins.

BACKGROUND: Barnacles are sessile crustaceans that attach to underwater surfaces using barnacle cement proteins. Barnacles have a calcareous or chitinous membranous base, and their substratum varies from biotic (e.g. corals/sponges) to abiotic surfaces. In this study, we tested the hypothesis that the cement protein (CP) composition and chemical properties of different species vary according to the attachment substrate and/or the basal structure. We examined the histological structure of cement glands and explored the variations in cement protein homologs of 12 barnacle species with different attachment habitats and base materials. RESULTS: Cement gland cells in the rocky shore barnacles Tetraclita japonica formosana and Amphibalanus amphitrite are eosinophilic, while others are basophilic. Transcriptome analyses recovered CP homologs from all species except the scleractinian coral barnacle Galkinia sp. A phylogenomic analysis based on sequences of CP homologs did not reflect a clear phylogenetic pattern in attachment substrates. In some species, certain CPs have a remarkable number of paralogous sequences, suggesting that major duplication events occurred in CP genes. The examined CPs across taxa show consistent bias toward particular sets of amino acid. However, the predicted isoelectric point (pI) and hydropathy are highly divergent. In some species, conserved regions are highly repetitive. CONCLUSIONS: Instead of developing specific cement proteins for different attachment substrata, barnacles attached to different substrata rely on a highly duplicated cementation genetic toolkit to generate paralogous CP sequences with diverse chemical and biochemical properties. This general CP cocktail might be the key genetic feature enabling barnacles to adapt to a wide variety of substrata.

Five hundred million years to mobility: directed locomotion and its ecological function in a turtle barnacle.

Movement is a fundamental characteristic of life, yet some invertebrate taxa, such as barnacles, permanently affix to a substratum as adults. Adult barnacles became 'sessile' over 500 Ma; however, we confirm that the epizoic sea turtle barnacle, Chelonibia testudinaria, has evolved the capacity for self-directed locomotion as adults. We also assess how these movements are affected by water currents and the distance between conspecifics. Finally, we microscopically examine the barnacle cement. Chelonibia testudinaria moved distances up to 78.6 mm yr-1 on loggerhead and green sea turtle hosts. Movements on live hosts and on acrylic panels occasionally involved abrupt course alterations of up to 90°. Our findings showed that barnacles tended to move directly against water flow and independent of nearby conspecifics. This suggests that these movements are not passively driven by external forces and instead are behaviourally directed. In addition, it indicates that these movements function primarily to facilitate feeding, not reproduction. While the mechanism enabling movement remained elusive, we observed that trails of cement bore signs of multi-layered, episodic secretion. We speculate that proximal causes of movement involve one or a combination of rapid shell growth, cement secretion coordinated with basal membrane lifting, and directed contraction of basal perimeter muscles.

A proteomic analysis of skeletal tissue anomaly in the brain coral Platygyra carnosa.

Coral skeletal growth anomaly (GA) is a common coral disease. It has been considered as a pathological condition comparable to abnormal tissue growth in mammals, but little is known about the molecular changes underlying coral GA. To investigate the molecular pathology of GA, we compared the proteome between normal and GA-affected tissues of the brain coral Platygyra carnosa using iTRAQ-labeling and LC-MS/MS, which quantified 818 proteins and identified 117 differentially expressed proteins (DEPs). GO analyses revealed DEPs that might be related to GA included "translational elongation", "proteasome core complex", "amine metabolic processes" and "lysosome". Several proteins implicated in calcification and fluorescence were differentially expressed at both protein and mRNA level. Protein-protein interaction network suggested possible involvement of TNF receptor signaling in GA. Overall, our results provided novel insights into the molecular pathology of coral GA, which will pave the way for determination of the causative agent(s) of this coral disease.

Exploring the Influence of Signal Molecules on Marine Biofilms Development.

Microbes respond to environmental stimuli through complicated signal transduction systems. In microbial biofilms, because of complex multiple species interactions, signals transduction systems are of an even higher complexity. Here, we performed a signal-molecule-treatment experiment to study the role of different signal molecules, including N-hexanoyl-L-homoserine lactone (C6-HSL), N-dodecanoyl-L-homoserine lactone (C12-HSL), Pseudomonas quinolone signal (PQS), and cyclic di-GMP (c-di-GMP), in the development of marine biofilms. Comparative metagenomics suggested a distinctive influence of these molecules on the microbial structure and function of multi-species biofilm communities in its developing stage. The PQS-treated biofilms shared the least similarity with the control and initial biofilms. The role of PQS in biofilm development was further explored experimentally with the strain Erythrobacter sp. HKB8 isolated from marine biofilms. Comparative transcriptomic analysis showed that 314 genes, such as those related to signal transduction and biofilm formation, were differentially expressed in the untreated and PQS-treated Erythrobacter sp. HKB8 biofilms. Our study demonstrated the different roles of signal molecules in marine biofilm development. In particular, the PQS-based signal transduction system, which is frequently detected in marine biofilms, may play an important role in regulating microbe-microbe interactions and the assemblage of biofilm communities.

Transcriptomic analysis of the mode of action of the candidate anti-fouling compound di(1H-indol-3-yl)methane (DIM) on a marine biofouling species, the bryozoan Bugula neritina.

Di(1H-indol-3-yl)methane (DIM) was previously suggested to be an environmentally friendly antifouling compound, but it was also reported that the compound was highly stable in natural seawater. The present study reported that 3 h DIM treatments at 4 µg mL-1 or higher concentration and 12 h DIM treatments at 2 µg mL-1 or higher concentration induced significant larval mortality and metamorphic abnormality in the bryozoan Bugula neritina. The bioassay results correlated with the dose-dependent up-regulation of HSP family proteins, pro-apoptotic proteins, ubiquitination protein, and the dose-dependent down-regulation of anti-apoptotic genes and developmental genes. Unexpectedly, genes involved in fatty acid biosynthesis and protein synthesis were up-regulated in response to DIM treatment, but, in general, the effects of DIM on B. neritina larvae were comparable to that reported in human cancer cell lines. DIM also induced changes in steroid hormone biosynthesis genes in B. neritina larvae, leading to the concern that DIM might have long-term effects on marine lives. Overall, the present study suggested that application of DIM to the bryozoan larvae would trigger a major transcriptomic response, which might be linked to the observed larval mortality and abnormality. We suggest that application of DIM as an antifouling ingredient should be proceeded with great cautions.

Exploring the regulatory role of nitric oxide (NO) and the NO-p38MAPK/cGMP pathway in larval settlement of the bryozoan Bugula neritina.

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.

HSP90 regulates larval settlement of the bryozoan Bugula neritina through the nitric oxide pathway.

The larvae of many sessile marine invertebrates go through a settlement process, during which planktonic larvae attach to a substrate and metamorphose into sessile juveniles. Larval attachment and metamorphosis (herein defined as 'settlement') are complex processes mediated by many signalling pathways. Nitric oxide (NO) signalling is one of the pathways that inhibits larval settlement in marine invertebrates across different phyla. NO is synthesized by NO synthase (NOS), which is a client of the molecular chaperone heat shock protein 90 (HSP90). In the present study, we provide evidence that NO, a gaseous messenger, regulates larval settlement of Bugula neritina By using pharmacological bioassays and western blotting, we demonstrated that NO inhibits larval settlement of B. neritina and that NO signals occur mainly in the sensory organ of swimming larvae. The settlement rate of B. neritina larvae decreased after heat shock treatment. Inhibition of HSP90 induced larval settlement, and attenuated the inhibition of NO donors during larval settlement. In addition, the expression level of both HSP90 and NOS declined upon settlement. These results demonstrate that HSP90 regulates the larval settlement of B. neritina by interacting with the NO pathway.

Exploring coral microbiome assemblages in the South China Sea.

Coral reefs are significant ecosystems. The ecological success of coral reefs relies on not only coral-algal symbiosis but also coral-microbial partnership. However, microbiome assemblages in the South China Sea corals remain largely unexplored. Here, we compared the microbiome assemblages of reef-building corals Galaxea (G. fascicularis) and Montipora (M. venosa, M. peltiformis, M. monasteriata) collected from five different locations in the South China Sea using massively-parallel sequencing of 16S rRNA gene and multivariate analysis. The results indicated that microbiome assemblages for each coral species were unique regardless of location and were different from the corresponding seawater. Host type appeared to drive the coral microbiome assemblages rather than location and seawater. Network analysis was employed to explore coral microbiome co-occurrence patterns, which revealed 61 and 80 co-occurring microbial species assembling the Galaxea and Montipora microbiomes, respectively. Most of these co-occurring microbial species were commonly found in corals and were inferred to play potential roles in host nutrient metabolism; carbon, nitrogen, sulfur cycles; host detoxification; and climate change. These findings suggest that the co-occurring microbial species explored might be essential to maintain the critical coral-microbial partnership. The present study provides new insights into coral microbiome assemblages in the South China Sea.

Metagenomic analysis reveals a green sulfur bacterium as a potential coral symbiont.

Coral reefs are ecologically significant habitats. Coral-algal symbiosis confers ecological success on coral reefs and coral-microbial symbiosis is also vital to coral reefs. However, current understanding of coral-microbial symbiosis on a genomic scale is largely unknown. Here we report a potential microbial symbiont in corals revealed by metagenomics-based genomic study. Microbial cells in coral were enriched for metagenomic analysis and a high-quality draft genome of "Candidatus Prosthecochloris korallensis" was recovered by metagenome assembly and genome binning. Phylogenetic analysis shows "Ca. P. korallensis" belongs to the Prosthecochloris clade and is clustered with two Prosthecochloris clones derived from Caribbean corals. Genomic analysis reveals "Ca. P. korallensis" has potentially important ecological functions including anoxygenic photosynthesis, carbon fixation via the reductive tricarboxylic acid (rTCA) cycle, nitrogen fixation, and sulfur oxidization. Core metabolic pathway analysis suggests "Ca. P. korallensis" is a green sulfur bacterium capable of photoautotrophy or mixotrophy. Potential host-microbial interaction reveals a symbiotic relationship: "Ca. P. korallensis" might provide organic and nitrogenous nutrients to its host and detoxify sulfide for the host; the host might provide "Ca. P. korallensis" with an anaerobic environment for survival, carbon dioxide and acetate for growth, and hydrogen sulfide as an electron donor for photosynthesis.

Molecular adaptation in the world's deepest-living animal: Insights from transcriptome sequencing of the hadal amphipod Hirondellea gigas.

The Challenger Deep in the Mariana Trench is the deepest point in the oceans of our planet. Understanding how animals adapt to this harsh environment characterized by high hydrostatic pressure, food limitation, dark and cold is of great scientific interest. Of the animals dwelling in the Challenger Deep, amphipods have been captured using baited traps. In this study, we sequenced the transcriptome of the amphipod Hirondellea gigas collected at a depth of 10,929 m from the East Pond of the Challenger Deep. Assembly of these sequences resulted in 133,041 contigs and 22,046 translated proteins. Functional annotation of these contigs was made using the go and kegg databases. Comparison of these translated proteins with those of four shallow-water amphipods revealed 10,731 gene families, of which 5659 were single-copy orthologs. Base substitution analysis on these single-copy orthologs showed that 62 genes are positively selected in H. gigas, including genes related to ß-alanine biosynthesis, energy metabolism and genetic information processing. For multiple-copy orthologous genes, gene family expansion analysis revealed that cold-inducible proteins (i.e., transcription factors II A and transcription elongation factor 1) as well as zinc finger domains are expanded in H. gigas. Overall, our results indicate that genetic adaptation to the hadal environment by H. gigas may be mediated by both gene family expansion and amino acid substitutions of specific proteins.

Genome Reduction and Microbe-Host Interactions Drive Adaptation of a Sulfur-Oxidizing Bacterium Associated with a Cold Seep Sponge.

As the most ancient metazoan, sponges have established close relationships with particular microbial symbionts. However, the characteristics and physiology of thioautotrophic symbionts in deep-sea sponges are largely unknown. Using a tailored "differential coverage binning" method on 22-Gb metagenomic sequences, we recovered the nearly complete genome of a sulfur-oxidizing bacterium (SOB) that dominates the microbiota of the cold seep sponge Suberites sp. Phylogenetic analyses suggested that this bacterium (an unclassified gammaproteobacterium termed "Gsub") may represent a new deep-sea SOB group. Microscopic observations suggest that Gsub is probably an extracellular symbiont. Gsub has complete sulfide oxidation and carbon fixation pathways, suggesting a chemoautotrophic lifestyle. Comparative genomics with other sponge-associated SOB and free-living SOB revealed significant genome reduction in Gsub, characterized by the loss of genes for carbohydrate metabolism, motility, DNA repair, and osmotic stress response. Intriguingly, this scenario of genome reduction is highly similar to those of the endosymbionts in deep-sea clams. However, Gsub has retained genes for phage defense and protein secretion, with the latter potentially playing a role in interactions with the sponge host. In addition, we recovered the genome of an ammonia-oxidizing archaeon (AOA), which may carry out ammonia oxidation and carbon fixation within the sponge body. IMPORTANCE Sponges and their symbionts are important players in the biogeochemical cycles of marine environments. As a unique habitat within marine ecosystems, cold seeps have received considerable interest in recent years. This study explores the lifestyle of a new symbiotic SOB in a cold seep sponge. The results demonstrate that both this sponge symbiont and endosymbionts in deep-sea clams employ similar strategies of genome reduction. However, this bacterium has retained unique functions for immunity and defense. Thus, the functional features are determined by both the symbiotic relationship and host type. Moreover, analyses of the genome of an AOA suggest that microbes play different roles in biochemical cycles in the sponge body. Our findings provide new insights into invertebrate-associated bacteria in cold seep environments.

In Silico Prediction of Neuropeptides/Peptide Hormone Transcripts in the Cheilostome Bryozoan Bugula neritina.

The bryozoan Bugula neritina has a biphasic life cycle that consists of a planktonic larval stage and a sessile juvenile/adult stage. The transition between these two stages is crucial for the development and recruitment of B. neritina. Metamorphosis in B. neritina is mediated by both the nervous system and the release of developmental signals. However, no research has been conducted to investigate the expression of neuropeptides (NP)/peptide hormones in B. neritina larvae. Here, we report a comprehensive study of the NP/peptide hormones in the marine bryozoan B. neritina based on in silico identification methods. We recovered 22 transcripts encompassing 11 NP/peptide hormone precursor transcript sequences. The transcript sequences of the 11 isolated NP precursors were validated by cDNA cloning using gene-specific primers. We also examined the expression of three peptide hormone precursor transcripts (BnFDSIG, BnILP1, BnGPB) in the coronate larvae of B. neritina, demonstrating their distinct expression patterns in the larvae. Overall, our findings serve as an important foundation for subsequent investigations of the peptidergic control of bryozoan larval behavior and settlement.

Characterization of Arginine Kinase in the Barnacle Amphibalanus Amphitrite and Its Role in the Larval Settlement.

Energy metabolism is a key process in larval settlement of barnacles, but the underlying molecular mechanisms remain ambiguous. Arginine kinase (AK) mainly participates in energy metabolism in invertebrates. So far, its roles in barnacles have not been studied. In the present study, we raised an antibody against AK from Amphibalanus amphitrite Darwin to characterize the roles of AK in the larval settlement process. Among the developmental stages, AK was highly expressed during the cypris stage. Along with the aging process in cyprids, the level of AK decreased. The immunostaining results showed that AK was localized to muscular tissues in cyprids, including antennules, antennular muscles, and thoracic limbs. The larval settlement rate decreased and larval movement was inhibited in response to treatments with high concentrations of AK inhibitors (rutin and quercetin). These results demonstrated that AK was involved in the larval settlement of A. amphitrite through mediating energy supply in muscle tissues. Moreover, further analysis indicated that both the p38 MAPK and NO/cGMP pathways positively mediated the expression of AK in cyprids.

High-throughput transcriptome sequencing of the cold seep mussel Bathymodiolus platifrons.

Bathymodiolid mussels dominate hydrothermal vents, cold methane/sulfide-hydrocarbon seeps, and other sites of organic enrichment. Here, we aimed to explore the innate immune system and detoxification mechanism of the deep sea mussel Bathymodiolus platifrons collected from a methane seep in the South China Sea. We sequenced the transcriptome of the mussels' gill, foot and mantle tissues and generated a transcriptomic database containing 96,683 transcript sequences. Based on GO and KEGG annotations, we reported transcripts that were related to the innate immune system, heavy metal detoxification and sulfide metabolic genes. Our in-depth analysis on the isoforms of peptidoglycan recognition protein (PGRP) that have different cellular location and potentially differential selectivity towards peptidoglycan (PGN) from gram-positive and gram-negative bacteria were differentially expressed in different tissues. We also reported a potentially novel form of metallothionein and the production of phytochelatin in B. platifrons, which has not been reported in any of its coastal relative Mytilus mussel species. Overall, the present study provided new insights into heavy metal and sulfide metabolism in B. platifrons and can be served as the basis for future molecular studies on host-symbiont interactions in cold seep mussels.

Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting.

Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/cGMP (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250, and 1000 µM) using a label-free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron-mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.

Chemical Component and Proteomic Study of the Amphibalanus (= Balanus) amphitrite Shell.

As typical biofoulers, barnacles possess hard shells and cause serious biofouling problems. In this study, we analyzed the protein component of the barnacle Amphibalanus (= Balanus) amphitrite shell using gel-based proteomics. The results revealed 52 proteins in the A. Amphitrite shell. Among them, 40 proteins were categorized into 11 functional groups based on KOG database, and the remaining 12 proteins were unknown. Besides the known proteins in barnacle shell (SIPC, carbonic anhydrase and acidic acid matrix protein), we also identified chorion peroxidase, C-type lectin-like domains, serine proteases and proteinase inhibitor proteins in the A. Amphitrite shell. The sequences of these proteins were characterized and their potential functions were discussed. Histology and DAPI staining revealed living cells in the shell, which might secrete the shell proteins identified in this study.

Synchronized dynamics of bacterial niche-specific functions during biofilm development in a cold seep brine pool.

The biology of biofilm in deep-sea environments is barely being explored. Here, biofilms were developed at the brine pool (characterized by limited carbon sources) and the normal bottom water adjacent to Thuwal cold seeps. Comparative metagenomics based on 50 Gb datasets identified polysaccharide degradation, nitrate reduction and proteolysis as enriched functional categories for brine biofilms. The genomes of two dominant species: a novel Deltaproteobacterium and a novel Epsilonproteobacterium in the brine biofilms were reconstructed. Despite rather small genome sizes, the Deltaproteobacterium possessed enhanced polysaccharide fermentation pathways, whereas the Epsilonproteobacterium was a versatile nitrogen reactor possessing nar, nap and nif gene clusters. These metabolic functions, together with specific regulatory and hypersaline-tolerant genes, made the two bacteria unique compared with their close relatives, including those from hydrothermal vents. Moreover, these functions were regulated by biofilm development, as both the abundance and the expression level of key functional genes were higher in later stage biofilms, and co-occurrences between the two dominant bacteria were demonstrated. Collectively, unique mechanisms were revealed: (i) polysaccharides fermentation, proteolysis interacted with nitrogen cycling to form a complex chain for energy generation, and (ii) remarkably exploiting and organizing niche-specific functions would be an important strategy for biofilm-dependent adaptation to the extreme conditions.

siRNA transfection in larvae of the barnacle Amphibalanus amphitrite.

RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in 'double transfections', in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments.

Pyrosequencing revealed shifts of prokaryotic communities between healthy and disease-like tissues of the Red Sea sponge Crella cyathophora.

Sponge diseases have been widely reported, yet the causal factors and major pathogenic microbes remain elusive. In this study, two individuals of the sponge Crella cyathophora in total that showed similar disease-like characteristics were collected from two different locations along the Red Sea coast separated by more than 30 kilometers. The disease-like parts of the two individuals were both covered by green surfaces, and the body size was much smaller compared with adjacent healthy regions. Here, using high-throughput pyrosequencing technology, we investigated the prokaryotic communities in healthy and disease-like sponge tissues as well as adjacent seawater. Microbes in healthy tissues belonged mainly to the Proteobacteria, Cyanobacteria and Bacteroidetes, and were much more diverse at the phylum level than reported previously. Interestingly, the disease-like tissues from the two sponge individuals underwent shifts of prokaryotic communities and were both enriched with a novel clade affiliated with the phylum Verrucomicrobia, implying its intimate connection with the disease-like Red Sea sponge C. cyathophora. Enrichment of the phylum Verrucomicrobia was also considered to be correlated with the presence of algae assemblages forming the green surface of the disease-like sponge tissues. This finding represents an interesting case of sponge disease and is valuable for further study.