Quantitative Proteomics Identify Molecular Targets That Are Crucial in

J. Proteome Res. , 2011, 10 (1), pp 349–360. DOI: 10.1021/pr100817v. Publication Date (Web): November 22, 2010. Copyright © 2010 American Chemical ...
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Quantitative Proteomics Identify Molecular Targets That Are Crucial in Larval Settlement and Metamorphosis of Bugula neritina Huoming Zhang,#,†,‡ Yue Him Wong,#,† Hao Wang,#,† Zhangfan Chen,† Shawn M. Arellano,† Timothy Ravasi,‡ and Pei-Yuan Qian*,† KAUST Global Collaborative Research Program, Section of Marine Ecology and Biotechnology, Division of Life Science, Hong Kong University of Science and Technology, Hong Kong SAR, China, and Red Sea Laboratory for Integrative Systems Biology, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia Received August 11, 2010

The marine invertebrate Bugula neritina has a biphasic life cycle that consists of a swimming larval stage and a sessile juvenile and adult stage. The attachment of larvae to the substratum and their subsequent metamorphosis have crucial ecological consequences. Despite many studies on this species, little is known about the molecular mechanism of these processes. Here, we report a comparative study of swimming larvae and metamorphosing individuals at 4 and 24 h postattachment using label-free quantitative proteomics. We identified more than 1100 proteins at each stage, 61 of which were differentially expressed. Specifically, proteins involved in energy metabolism and structural molecules were generally down-regulated, whereas proteins involved in transcription and translation, the extracellular matrix, and calcification were strongly up-regulated during metamorphosis. Many tightly regulated novel proteins were also identified. Subsequent analysis of the temporal and spatial expressions of some of the proteins and an assay of their functions indicated that they may have key roles in metamorphosis of B. neritina. These findings not only provide molecular evidence with which to elucidate the substantial changes in morphology and physiology that occur during larval attachment and metamorphosis but also identify potential targets for antifouling treatment. Keywords: Biofouling • Bugula neritina • larval attachment and metamorphosis • label-free quantitative proteomics

1. Introduction Many sessile marine invertebrates have a biphasic life cycle that consists of a swimming larval stage and juvenile and adult stages that are attached to a submerged surface.1 The transition between the swimming and sessile phases involves larval attachment and metamorphosis (often referred as larval settlement). Larval attachment and early metamorphosis are critical to the survival of the species and the structures of communities and have important economic, as well as ecological, consequences.2,3 It has been estimated that the undesirable settlement of the larvae of these invertebrates on commercially important marine structures such as aquaculture nets, immersed pipelines and ship hulls costs over US$ 6.5 billion per year worldwide.4 However, an effective and environmentally friendly antifouling treatment is not yet available, largely due to a lack of information about the molecular mechanism of larval settlement. The cheilostome bryozoan Bugula neritina (Linne´) is one of the most abundant fouling organisms.5 It is widely distributed * Corresponding author. Tel: (+852) 2358 7331. Fax: (+852) 2358 1559. E-mail: [email protected]. # These authors contributed equally. † Hong Kong University of Science and Technology. ‡ King Abdullah University of Science and Technology. 10.1021/pr100817v

 2011 American Chemical Society

in subtropical and temperate marine waters.6 B. neritina larvae newly released from adults are competent to metamorphose within minutes without the need of an inducer.7 This metamorphosis can be divided into two phases.7 The first phase is drastic and quick, and is initiated when the swimming larvae attach to the substratum, with concomitant morphogenetic movements including the eversion of the internal sac, the retraction of the apical disk, and the involution of the corona to form the precursor to the cystid and polypide.8 The second phase of metamorphosis is more gradual, ranging in duration from 36 to 48 h, and includes the complete degradation of the larval tissues. Although B. neritina metamorphosis is wellunderstood in terms of morphological, physiological, structural, and functional changes,9 the key molecules that govern the underlying mechanism remain unclear. Comparative analysis of the gene or protein expression profiles at different developmental stages could thus identify differential or stage-specific expression during larval metamorphosis and aid the discovery of novel targets for antifouling treatment. The study of protein expression in whole organisms during their development or therapeutic intervention poses great challenges due to their complexity and the lack of genetic information on nonmodel organisms. An earlier study using traditional two-dimensional gel electrophoresis (2-DE) preJournal of Proteome Research 2011, 10, 349–360 349 Published on Web 11/22/2010

research articles sented about 400 spots and revealed the pattern changes during barnacle metamorphosis.10 The use of sample prefractionation, narrow pI and sensitive fluorescent stain for 2-DE enhanced the resolution and increased the number of protein spots by ∼1.5-fold.11,12 However, only relatively abundant protein spots were identified using mass spectrometry from these studies, suggesting that an alternative gel-free proteomics technique may be required for the identification of lower abundance but important protein markers in metamorphosis of marine invertebrates. Recently, we profiled the transcriptome of B. neritina and generated a large number of ESTs,13 making it possible to perform high throughput gel-free proteomics study. In this study, we analyzed the proteomes of B. neritina of three early developmental stages using label-free quantitative proteomics. Sixty-one proteins were differentially expressed and collagens were confirmed to play key roles in metamorphosis of B. neritina.

2. Materials and Methods 2.1. Preparation of Larvae. Adult Bugula neritina colonies were collected from a fish-farm (22°21′19.42′′N, 114°16′14.78′′E) in San Shing Wan, Hong Kong, in November 2009. They were maintained in a flow-through seawater system in our laboratory and used within seven days. To induce release of larvae, the adult B. neritina colonies were transferred into a 10-L glass tank with seawater filtered with a 0.45-µm filter (Millipore, Bedford, MA, USA) and then exposed to a bright artificial light. The released larvae were carefully selected under a dissecting microscope to avoid contamination. Approximately 15 000 swimming larvae were equally divided into three groups: swimming larvae, 4 h postattachment larvae, and 24 h postattachment larvae. For the swimming larvae group, the larvae were collected upon release on a mesh sieve and transferred to a 2-mL Eppendorf tube. These newly released larvae (swimming larvae) are competent to attach and metamorphose within minutes in the dark without the need of an inducer. They were quickly washed with autoclaved filtered seawater (AFSW) and stored at -80 °C. For the 4 and 24 h postattachment groups, the swimming larvae were transferred to Petri dishes containing 10 mL of AFSW and allowed to attach and metamorphose in the dark for 30 min, after which unattached larvae were discarded. Attached individuals were allowed to metamorphose for 4 and 24 h, respectively, before being scraped off the Petri dish with a sterile razor blade. These samples were then collected, washed, and stored at -80 °C as per the procedure for swimming larvae. 2.2. Protein Extraction and Digestion. The larval samples were resuspended in 1 mL of 1% SDS solution supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). They were homogenized using Dounce homogenizer and sonicated intermittently using a Misonix sonicator-XL2020 (Misonix, Farmingdale, NY, USA). The unlysed larvae and debris were pelleted down by centrifugation at 15000g at 4 °C for 10 min. The supernatants containing the larval proteins were transferred into new tubes and the proteins were purified by acetone precipitation (1:6 sample/acetone v/v) at -20 °C, overnight. The protein pellets were resuspended in 8 M of urea solution and the contents were quantified using an RC-DC kit (Bio-Rad, Hercules, CA, USA). Approximately 1.5 mg of proteins were used for each in-solution digestion.14,15 Briefly, the proteins were reduced with 10 mM of dithiothreitol at 37 °C for 30 min and alkylated with 40 mM of iodoacetamide for 1 h in the dark at room temperature (RT). They were then diluted 350

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Zhang et al. 7-fold with 50 mM of NH4HCO3 before digestion with trypsin (Promega, Madison, WI, USA) overnight at 37 °C in a 1:50 trypsin-to-protein mass ratio. The protein digest was desalted using Sep-Pak C18 cartridges (Waters, Milford, MA, USA) and dried in a SpeedVac (Thermo Electron, Waltham, MA, USA). 2.3. LC-MS Analysis. The dried digest was reconstituted and fractionated by strong cation exchange chromatography.16 Twenty fractions were obtained for mass spectrometry analysis. Each of the factions was reconstituted in 10 µL of 0.1% formic acid. The samples were concentrated in a peptide trap (Waters) and then analyzed using a nanoflow UPLC (nanoAcquity, Waters) coupled with an ESI-hybrid Q-TOF (Premier, Waters) tandem mass spectrometer. The peptide separation was performed in a BEH130 C18 analytical column (75 µm inner diameter × 25 cm, 1.7-µm particles, Waters). Mobile phase A (0.1% formic acid in H2O) and mobile phase B (0.1% formic acid in acetonitrile) were used to establish a 120-min gradient comprising 3 min at 5% B, 70 min of 5-30% B, 22 min of 30-80% B; maintenance 10 min at 80% B, and re-equilibration 15 min at 5% B. The UPLC was operated at a constant flow rate of 0.2 µL/min. The ion source was set as a capillary voltage of 2.4 kV, a source temperature of 80 °C, a sample cone voltage of 35 V, and a collision cell gas flow rate of 0.5 mL/min. The lock mass was sampled every 30 s. The Q-TOF was set to perform data-dependent acquisition in the positive ion mode with a selected MS survey mass range of 300-1600 m/z. The three most abundant peptides with +2 to +4 charge states above a 40-count threshold were selected for MS/MS. The time of summation of the MS/MS events was set to 3 s, with dynamic exclusion for 60 s with a (30 mDa mass tolerance. 2.4. Database Search and Data Analysis. The raw MS data were converted into peak list files in pkl format using Proteinlynx (version 2.2.5, Waters) (smooth 3/2 Savitzky Golay and center 4 channels/80% centroid) and merged using Mascot daemon (version 2.2) before running a Mascot search against a customized B. neritina database.13 The enzyme limits were set at full tryptic cleavage at both ends, and a maximum of two missed cleavages were allowed. The mass tolerances were set 20 ppm for the peptide precursors and 0.5 Da for the fragment ions. Carboxamidomethylation (+57.02) at cysteine residues was set as fixed modification, and oxidation (+15.99) at methionine, phosphorylation at serine, threonine, or tyrosine (+79.96) was set as variable modifications. The resulting .dat files from the Mascot search were processed using PeptideProphet17and ProteinProphet,18 which are part of the Trans-Proteomic Pipeline. The false positive rate in each experiment was controlled to 95% accuracy was achieved when a >1.5-fold change was used as the threshold to measure significant changes in a triplicate experiment. This accuracy increased to 98.8% when only the relatively high abundance proteins (>5000 molecules/cell) were included in the calculation (Table S4, Supporting Information). We also analyzed the nine LC-MS experiments using principal component analysis (PCA). PCA reflects the similarity among different samples or replicates. As shown in Figure 1, the scores for each stage tended to form clusters, whereas those for different stages separated along the first two axes of major variation. We then compared the proteome expression profiles of the three developmental stages to identify differential expression or stage-specific proteins. As discussed, a 1.5-fold change was used as the cutoff, and only proteins with >5000 molecules per cell in at least one stage were taken into consideration, and protein abundance from biological replicates had to demonstrate a 4% of the total protein abundance at each stage. This protein was upregulated by 2-fold in the 4 and 24 h postattachment larvae. Although the exact function of this protein remains to be characterized, bioinformatic analysis using its full-length amino acid sequence revealed that it belongs to the extracellular matrix and thus probably has an important role in larval attachment and metamorphosis. 3.2.5. Functionally Ungrouped Proteins. In addition to the aforementioned functional groups, we identified many proteins that were either of unknown function(s) or that did not belong to any known functional group. The expressions of many of these proteins have been shown in other species to be related to development. For example, the carbonic anhydrase II (CA II) detected at 24 h post attachment has been found to be substantially up-regulated in the metamorphosis of various coral species.42-44 The CA proteins are involved in pH and CO2/ bicarbonate homeostasis and play a crucial role in the calcification process. The deposition of calcium carbonate in an organic matrix45 is initiated immediately after attachment46 and forms strong exoskeletons during late metamorphosis. The inhibition of CA activity results in retarded shell formation in the barnacle Balanus improvisus47 and the mussel Unio pictorum,48 leading to the concomitant inhibition of growth and Journal of Proteome Research • Vol. 10, No. 1, 2011 355

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Figure 2. Western blot confirmation of the differential expression of two cytoskeleton proteins. Tubulin was down-regulated after attachment (both the 4 and 24 h postattachment stages), whereas actin was down-regulated at the 4 h postattachment stage but up-regulated at the 24 h postattachment stage. GAPDH was confirmed to have a stable expression and was thus used as the loading control.

development. A similar role in B. neritina can thus be expected. Indeed, although shell formation does not occur, the calcification of the body wall in B. neritina is required from an advanced preancerstrula stage onward.9 Translationally controlled tumor protein (TCTP) is a highly conserved protein and is upregulated in various tumors. Reducing TCTP levels in Drosophila reduces cell size, cell number, and organ size.49 The upregulation of TCTP protein may be important for the organ development during metamorphosis of B. neritina. Cathepsin L and leukotriene A-4 hydrolase are important for the overall degradation of proteins. Their up-regulation may be necessary during metamorphosis in which high rates of protein synthesis and degradation are involved in concert with drastic morphological rearrangement. Several calcium-binding proteins, including calcium/calmodulin-dependent protein kinase, calmodulin, and a neuronal acetylcholine receptor, were upregulated in the 4 h postattachment stage. In the first phase of metamorphosis, morphological rearrangement is mediated by larval muscle contraction9 and possibly the nervous system.50 The nervous system may be responsible for perceiving the stimulants of attachment, and for initiating and coordinating the subsequent larval muscle contraction during the initial phase of metamorphosis, and thus necessitating the upregulation of calcium-binding proteins. Temptin was up-regulated at the 4 h postattachment stage. In marine mollusk Aplysia, temptin is a water-borne protein pheromone and functions to attract potential mates to form and maintain breeding aggregations.51 A similar role could be expected in B. neritina. In addition to the functionally annotated proteins, more than 10 novel proteins were differentially expressed during metamorphosis in this study. Further studies of the functions of these proteins could lead to the discovery of their roles in regulating larval settlement. 3.3. Western Blot Confirmation and Real-Time PCR Analysis. To confirm our proteomics data, we performed a Western blot for three proteins for which antibodies are commercially available. In our data, GAPDH was found to be constantly expressed and was thus used as the loading control. As shown in Figure 2, the Western blots showed similar expression profiles to those from our proteomics analysis: tubulin-beta was down-regulated >1.5 fold at both the 4 and 24 h postattachment stages, whereas the expression of actin was decreased at the 4 h postattachment stage and then increased at the 24 h postattachment stage. These results provide evidence for the accuracy of our quantitative proteomics. 356

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Zhang et al. For most of the proteins identified from B. neritina, there is no commercial antibody. We thus used semiquantitative RTPCR to analyze the gene expressions of selected targets. As shown in Table 1, there was a positive correlation between the transcriptional and translational expression profiles in approximately 50% of the tested targets, whereas less than 20% of the genes showed the reversed expression trend. This result is consistent with similar measurements of gene and protein expression52 and can be attributed to post-transcriptional regulatory mechanisms. Notably, the gene expression of several proteins classified as being involved in the extracellular matrix increased very significantly. Of these, two collagens showed a >50-fold increase at the 4 h postattachment stage and a >500fold increase at the 24 h postattachment stage, whereas hypothetical protein (Contig24_3) showed a 2-fold increase at the 4 h postattachment stage and a 36-fold increase at the 24 h postattachment stage. The drastic increase in both protein and gene expression levels strongly indicates the important role of these genes in regulating larval metamorphosis. 3.4. Localization of Differentially Expressed Genes. We next examined the specific spatial expressions of several important genes and compared their expressions in the swimming and 4 h postattachment larvae using whole mount in situ hybridization (ISH). The ISH for the 24 h postattachment stage was not successful, probably due to the inaccessibility of the calcified larval body to probes, and the results are not shown here. As shown in Figure 3A, carbonic anhydrase was not detected at the swimming stage but was mainly expressed in the epidermal cells of the basal part of the 4 h postattachment larvae (Figure 3B). This expression pattern correlates well with an earlier observation that body wall calcification begins near the base of the complete ancestrula during the second stage of metamorphosis,9 and thus suggests that increased expression and localization of carbonic anhydrase in the epidermal cells may be essential for the calcification of the ancestrula in the later stage of metamorphosis. Antistasin indirectly regulates cytoskeleton structures and inhibits various proteases.53 It was strongly expressed in the lower wall region of the internal sac (Figure 3C). After the involution of the internal sac, the wall region becomes the precursor of the cystid and forms a new epithelium at the 4 h postattachment larvae (Figure 3D). We suggest the role of antistasin is to coordinate the formation of the new epithelium during the first phase of metamorphosis. Two histone transcripts (Contig2276_4 and Contig4643_2) showed a similar expression pattern in the interior of the 4 h postattachment larvae (Figure 3F,H). However, histone H2A.x (Contig4643_2) was not detected at the swimming stage, but histone H1 (Contig2276_4) was identified in the blastemal layers, which consist of a mass of undifferentiated cells. Following morphogenetic rearrangement in the first phase of metamorphosis, the blastemal layers are pulled into the interior of the preancestrula.8 Thus, it is probably the case that the expression of the two histone transcripts remains in the blastemal layer during metamorphosis from the swimming larvae to 4 h postattachment. The blastemal layers are the precursor of the lophophore and the digestive system, and differentiation of these organs occurs in late metamorphosis.9 Thus, the accumulation of histones within the blastemal layers is necessary for the preparation of organ development in late metamorphosis that requires active transcription and translation. Hypothetical protein (Contig1179_10) was exclusively expressed in the neck region of the internal sac in the swimming larvae (Figure 3I). The neck region contains highly

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Figure 3. Localization of gene expression by in situ hybridization. Carbonic anhydrase was not at the swimming larvae (A) but was expressed (purple color stain as shown by arrow) at the basal part of the 4 h postattachment larvae (B); antistasin was localized in the lower wall region of the internal sac of the swimming larvae (C) and in the epidermal cells of the basal part of the 4 h postattachment larvae (D); histone H1 (Contig2276_4) and histone H2A.x (Contig4643_2) showed a similar expression pattern in the interior of the 4 h postattachment larvae (F and H), whereas at the swimming stage, histone H1 was localized in the blastemal layers (E) and histone H2A.x was not detected (G). The hypothetical protein (Contig1179_10) was exclusively expressed in the neck region of the internal sac at the swimming larvae (I) but was absent at the 4 h postattachment stage; temptin was localized in the upper wall region of the internal sac of the swimming larvae (K) and the epidermal cells of the basal part of the 4 h postattachment larvae (L); negative controls for the swimming larvae (M) and the 4 h postattachment larvae (N). Star (*) indicates the aboral side of the swimming larvae.

columnar cells and abundant inclusion bodies, which secrete adhesive for cementing the animal to the substratum in the first phase of metamorphosis. The specific localization of this hypothetical protein may thus indicate that it has either a role in assisting the secretion of cement protein from the neck region during larval attachment or functions as a cement protein by itself. Consistent with this, bioinformatic analysis revealed that this protein contains a signal peptide and has functions in cellular macromolecule metabolism and biopolymer biosynthesis. The ISH revealed that temptin was expressed in the upper wall region of the internal sac of the swimming larvae and the epidermal cells of the basal part of the 4 h postattachment larvae (Figure 3K,L). In the larval attachment experiments, we frequently observed that the swimming larvae preferred to attach to the basal part of the preancestrula, which suggests that temptin may act as a con-specific attracting cue for larval attachment. Collectively, the localization of these differentially expressed transcripts in the important organs clearly suggested their critical roles in the regulation of larval attachment and metamorphosis. 3.5. Functional Assay Using a Collagen Inhibitor. As both the gene and protein expression levels of collagens were substantially increased during metamorphosis, we hypothesized that these proteins have an essential role in metamorphosis and that inhibition of their expression would affect larval growth and development. To test this hypothesis, we treated

larvae with a collagen inhibitor at different concentrations. We found that the larvae were able to attach to the substratum and initiate metamorphosis under all of the concentrations tested. The morphology and larval length at 30 min postattachment showed no difference between the treated and control groups. However, at the 4 h postattachment stage, the treated larvae showed retarded growth of the tubular body as measured by a shorter body length. Larval metamorphosis was affected by the collagen inhibitor at concentrations as low as ∼3 µM. Larval elongation was observed to be severely affected and retarded at concentrations greater than ∼12 µM with growth nearly ceasing between the 4 and 24 h postattachment stages (Figure 4B) and abnormal morphology compared with the controls (Figure 4A). Collagens have been reported to play an important role in the settlement of gemmules to the substratum and their subsequent morphogenesis.54 The inhibition of collagen synthesis prevents the settlement of gemmules and results in incomplete morphogenesis.55 Collagen IV is critical for forming a stable basement membrane and for providing flexibility and variability during cell proliferation.56 Collagens are also known to be major proteins in the extracellular matrix. The deposition of calcium carbonate to the extracellular matrix is required for larval metamorphosis,46 probably due to the need for mechanical strength to support the altered morphology. Interestingly, our results showed that collagens are not required for the larval Journal of Proteome Research • Vol. 10, No. 1, 2011 357

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Figure 4. Effect of a collagen inhibitor on larval metamorphosis. The collagen inhibitor did not affect the larval attachment, but severely affected larval metamorphosis at concentrations as low as ∼12 µM. (A) Light micrographs of B. neritina larvae at different developmental stages: the swimming larvae (I) immediately after release from the adult, and metamorphosing individuals at 30 min postattachment (II), 4 h postattachment (III), and 24 h postattachment (IV). Scale bar ) 100 µm. (B) Quantitative measurement of the elongation of metamorphosing B. neritina.

attachment but are crucial for the subsequent larval metamorphosis in B. neritina, indicating a possible taxonomic difference for antifouling relevant applications. 3.6. Implication for Antifouling Application. Our proteomics study identified a set of proteins that are differentially expressed during larval attachment and metamorphosis. Inhibiting/intervention of the expression of these proteins could disturb the larval settlement process and thus has a potential for antifouling application. Indeed, inhibition of collagen expression has been demonstrated to severely hinder larval metamorphosis, although it did not affect larval attachment in B neritina. As a result, the formation of adult colony or community of B neritina may be effectively prevented. Similar effect of targeting several other proteins could also be expected. 358

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For instance, proteins involved in shell formation (carbonate anhydrase) and con-specific attraction (the pheromone temptin) during metamorphosis could have a similar implication on antifoulant development. In fact, pheromone interference has been suggested to be an environmentally friendly antifouling strategy.57,58 Further study on the molecular regulation of these proteins in B. neritina may assist the development of an effective antifouling strategy.

4. Conclusion Analysis of B. neritina larvae by label-free quantitative proteomics has enabled us to present the first relatively comprehensive report of the expression patterns of the three developmental stages of this species. Comparison of the

Molecular Targets in B. neritina proteomes led to the identification of 61 differentially expressed proteins. Functional analysis of these proteins revealed the overall down-regulation of proteins involved in energy metabolism and structure, increased actin and tubulin depolymerization in the first phase of metamorphosis, and the significant up-regulation of transcription and translation proteins during subsequent larval development. These results provide molecular evidence to elucidate the substantial changes in morphology, physiology, structure, and function that occur during larval attachment and metamorphosis. We also identified many key molecules, such as histones, collagens, carbonic anhydrase, TCTP, and pheromone temptin. The subsequent localization of certain targets in the important structures actively involved in metamorphosis strongly indicates that they have a critical role in regulating the larval metamorphosis of B. neritina. Further functional assays and characterization of the candidates identified by this study should provide an ideal opportunity to uncover the detailed mechanism of larval attachment and metamorphosis in B. neritina, and possibly other marine fouling organisms.

Acknowledgment. We thank Jin Sun for his invaluable discussion and Cherry Hoi Ting Kwan for her comments on the manuscript. This study was supported by award SA-C0040/UK-C0016 from the King Abdullah University of Science and Technology, and grants (N-HKUST602/09 662408 and AoE/P-04/04-II) from the Research Grants Council of the Hong Kong Special Administrative Region to P.Y.Q. Supporting Information Available: Table S1: primer sequences used for real-time PCR in this study. Table S2: primer sequences used for gene cloning and in situ hybridization. Table S3: total proteins identified and quantified from each of the nine LC-MS experiments. Table S4: determination of the optimal cutoff for measurement of significant changes. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Qian, P. Y. Larval settlement of polychaetes. Hydrobiologia 1999, 402, 239–253. (2) Wahl, M. Marine epibiosis. I. Fouling and antifouling: some basic aspects. Mar. Ecol.: Prog. Ser. 1989, 58 (1), 175–189. (3) Lau, S. C. K.; Qian, P. Y. Inhibitory effect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite Darwin. Biofouling 2000, 16 (1), 47–58. (4) Zapata, M.; Silva, F.; Luza, Y.; Wilkens, M.; Riquelme, C. The inhibitory effect of biofilms produced by wild bacterial isolates to the larval settlement of the fouling ascidia Ciona intestinalis and Pyura praeputialis. Electron. J. Biotechnol. 2007, 10, 149–159. (5) Dahms, H. U.; Dobretsov, S.; Qian, P. Y. The effect of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina. J. Exp. Mar. Biol. Ecol. 2004, 313 (1), 191–209. (6) Ryland, J. S. Bugula simplex Hincks, a newly recognized polyzoan from British waters. Nature 1958, 181, 1148–1149. (7) Wong, Y. H.; Arellano, S. M.; Zhang, H.; Ravasi, T.; Qian, P. Y. Dependency on de novo protein synthesis and proteomic changes during metamorphosis of the marine bryozoan Bugula neritina. Proteome Sci. 2010, 8, 25. (8) Reed, C. G.; Woollacott, R. M. Mechanisms of rapid morphogenetic movements in the metamorphosis of the bryozoan Bugula neritina (Cheilostomata, Cellulariodea). I. Attachment to the substratum. J. Morphol. 1982, 172, 335–348. (9) Woollacott, R. M.; Zimmer, R. L. Attachment and metamorphosis of the cheilo-ctenostome bryozoan Bugula neritina (Linne). J. Morphol. 1971, 134 (3), 351–382. (10) Thiyagarajan, V.; Qian, P. Y. Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle Balanus amphitrite. Proteomics 2008, 8 (15), 3164–3172.

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