Transcriptome and proteome dynamics in larvae of the barnacle Balanus Amphitrite from the Red Sea

The barnacle Balanus amphitrite is widely distributed in marine shallow and tidal waters, and has significant economic and ecological importance. Nauplii, the first larval stage of most crustaceans, are extremely abundant in the marine zooplankton. However, a lack of genome information has hindered elucidation of the molecular mechanisms of development, settlement and survival strategies in extreme marine environments. We sequenced and constructed the genome dataset for nauplii to obtain comprehensive larval genetic information. We also investigated iTRAQ-based protein expression patterns to reveal the molecular basis of nauplii development, and to gain information on larval survival strategies in the Red Sea marine environment. A nauplii larval transcript dataset, containing 92,117 predicted open reading frames (ORFs), was constructed and used as a reference for the proteome analysis. Genes related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. We observed remarkable plasticity in the proteome of Red Sea larvae. The proteins associated with development, stress responses and osmoregulation showed the most significant differences between the two larval populations studied. The synergistic overexpression of heat shock and osmoregulatory proteins may facilitate larval survival in intertidal habitats or in extreme environments. We presented, for the first time, comprehensive transcriptome and proteome datasets for Red Sea nauplii. The datasets provide a foundation for future investigations focused on the survival mechanisms of other crustaceans in extreme marine environments.


Background
Barnacles are marine dominant fouling organisms accumulate on ship hulls, piers and under water structures; biofouling by these invertebrates results in an increase in power and fuel consumption [1][2][3] associated with marine transport. Removing biofouling barnacles is a major expense involving significant economic losses in shipping and other marine industries. However, some barnacles are of great commercial importance; for example, the gooseneck barnacle achieves an average market price of 30-60 USD per kilogram, depending on the season [4]. Such species are highly exploited, involving harvesting of approximately 500 kg per season in Spain and Portugal, and hence there is an urgent need for strategies to protect the stocks. The barnacle Balanus amphitrite is a widely distributed dominant fouling organism and a major component of rocky shore benthic communities [5][6][7]. Therefore, B. amphitrite is an excellent model organism for research on settlement biology, adaptation and biofouling. Nauplii are the earliest free-swimming stage in the development of most crustaceans, including barnacles [8], and are extremely abundant in the marine ecosystems.
Barnacle embryos are brooded by the parents, and the nauplii are released into the water column as swimming larvae [9]. The nauplius I stage has an elongated and pearshaped body, and the abdominal and caudal spines are partially differentiated. The eye is located in a front medial position and occurs in all developmental stages. The presence of posteriorly folded frontal horns and simple setae on the limbs are characteristics of the nauplius I stage. The newly released larvae swim actively and molt to the next developmental stage (nauplius II) within 30 min [10]. In the nauplius II stage the carapace extends in all directions and the entire body develops a bell shape that has numerous small spines and a pair of prominent spines. The nauplius II molts to the nauplius III stage in 38 h [11]. The nauplius III stage undergoes several moults before it transforms into a cyprid stage [12]. Molecular studies defining the role of gene or protein expression signatures in development and adaptive strategies of marine larvae in the natural marine environment are limited. Previous studies have used a broad range of proteomics and genomic tools to identify proteins and genes in larval stages of barnacles. However, these studies have mainly focused on exploring settlement and attachment processes. For example, Chen et al. (2011) identified 7954 putative differentially-expressed genes in larvae and adults of B. amphitrite using 454 pyrosequencing [13], and Yan et al. (2012) carried out in silico transcriptome data mining to study neuropeptides and their possible functional role in barnacle larval settlement [14]. Similarly, De Gregoris et al. (2012) generated deep sequencing EST libraries for nauplii and the cyprid and adult stages of B. amphitrite and identified unique contigs for each developmental stage [15]. More recently, Lin et al. (2014) performed Illumina sequencing of a membranous-based barnacle and identified cement-and adhesion-related genes [16]. Thiyagarajan and Qian (2008) reported the first study of global proteome expression patterns in cyprids and metamorphosed larvae of B. amphitrite [17], and  identified proteins from cyprid larvae using prefractionation methods to reduce sample complexity [18].  used multiplex proteomics to describe a distinct protein glycosylation pattern during barnacle larval metamorphosis [19], and Chen et al. (2014) recently used label-free quantitative proteomics to identify 700 proteins from nauplii II and VI, cyprids, and juvenile stages of B. amphitrite [20]. In addition, several studies have demonstrated the feasibility of using proteomics tools to investigate the mode of action of antifoulant chemicals in preventing barnacle larval settlement [21][22][23]. These studies have provided a wealth of molecular information, but comprehensive genome and proteome datasets for nauplius larvae are still lacking. Thus, in the present study we revealed sequence information and investigated the dynamics of protein expression patterns of nauplius larvae. Patterns of differential protein expression between two larval populations from contrasting marine environments were screened to investigate the survival strategies of larvae. Extreme marine environments pose great challenges for the survival of marine larvae. For example, the Red Sea (RS) is one of the warmest, most saline bodies of water; it receives little rainfall and has reduced oxygen solubility and high evaporation rates [24,25]. The ranges of salinity (36-41psu) and temperature (30-33°C) in the RS are considered extreme. In contrast, in Hong Kong (HK) marine water the salinity and temperature ranges are 28-31psu and 26-28°C, respectively [26,27]. The RS has a low concentration of dissolved oxygen (4.3-5.6 mg/L) compared with that of HK marine water (6.1-7.3 mg/L). Therefore, we hypothesized that changes in protein expression patterns would reflect the adaptation of barnacle larvae to the RS marine environment. To test this hypothesis we adopted a population proteomics approach to compare protein expression in larvae from HK marine waters and the RS. The specific objectives were: (i) to generate a comprehensive transcriptome dataset for the nauplius larvae of B. amphitrite; (ii) to compare the proteomes of barnacle larvae populations from the RS and HK; and (iii) to explore survival strategies of larvae in the RS environment. Figure 1 shows the experimental design used for the transcriptome and proteome analyses. Sequencing data statistics, assembly and annotation information for newly released nauplii of RS B. amphitrite are shown in Table 1 contigs. In total, 76,263 contigs showed at least one gene ontology (GO) annotation while 403,659 contigs remained hypothetical, and 33,992 contigs showed at least one Reactome annotation, whereas no annotation was found for 445,930 contigs. A total of 1,613,892 were assigned to 'biological process' (BP), and 508,916 and 664,282 contigs were assigned to 'molecular function' (MP) and 'cellular component' (CC), respectively (Additional file 1: Table S1-S4). Figure 2 shows the top 10 categories of assembled contigs belonging to the BP, MF and CC categories. The GOs having the top 10 hits and contigs under the BP category were 'embryo development' (14091), 'transport' (11260), 'reproduction' (11047), 'transcription regulation' (10440), 'larval development' (10417), 'signal transduction' (10150), 'metabolic process' (9767), 'small molecule metabolic process' (9316), 'transcription' (8757) and 'locomotion' (8743).

Abundant transcripts, protein domains and Reactome pathways
Functional analysis of contigs revealed that transcripts related to translation, oxidative phosphorylation and cytoskeletal development were highly abundant. The top 40 abundant transcripts and the number of reads  Contigs with at least one GO annontation 76,263 Contigs with at least one REACTOME annontation 33,992 are shown in Additional file 2: Table S5. Elongation factor-1 alpha was ranked first (1,698,408 reads), followed by NADH dehydrogenase subunit 4 (1,100,166 reads). Notably, four transcripts encoding cytochrome oxidase subunit I were also well represented. Interestingly, 10 of the 40 most abundant transcripts encoded hypothetical or predicted proteins. Additional file 3: Table S6 lists the top 30 Pfam domains predicted in the transcriptome. We were able to map 26 pathways present in the Reactome database (Additional file 4: Table S7). These pathways were highly overlapping in terms of the GO BP ( Fig. 2, Additional file 1: Table S2) and differentiallyexpressed protein categories (Table 2). A large number of genes related to larval development were found to be expressed (Fig. 3a), among which those for brain (GO:0007420; 3884 contigs), muscle (GO:0007517; 2826 contigs), eye (GO:0048749; 1842 contigs), digestive track (GO:0048565; 722 contigs) and cuticle (GO:0040003; 652 contigs) were dominant. Notably, 491 genes related to metamorphosis (GO:0007552) were also identified. A substantial number of genes assigned to signaling pathways involved in larvae development were also detected ( Fig. 3b). We found that a large number of genes involved in stress and osmoregulation were overrepresented. Genes expressed in response to high salinity, high temperature, cellular stress and ultraviolet (UV) light exposure were identified (Fig. 4a). We also identified several genes involved in homeostasis (Fig. 4b).
The larvae proteome A total of 1264 proteins from HK and RS nauplii were identified based on 4434 unique peptides and 5286 unique spectra (Additional file 5: Table S8). Proteins were quantified using HK larvae as a reference (reporter ion intensities were adjusted to 1; fold change > 1.5). Among these proteins, 170 showed differential expression in RS larvae, based on an average of 1.9 unique peptides, 2.22 spectra and 22.76 % sequence coverage. Of these proteins, 89 were up-regulated and 81 were down-regulated in RS larvae (Tables 2 and 3). Additional file 6: Table S9, Additional file 7: Table S10 and Additional file 8: Table S11 shows the number of peptides and the spectra, sequence coverage, peptide sequences and fold changes for each differential protein.

Differences in proteome expression between RS and HK larvae
Principle component analysis (PCA) of the proteome datasets showed clear separation of the HK and RS samples (Fig. 5), and hierarchical clustering analysis revealed two clusters of down-regulated (green) and up-regulated (red) proteins in the RS larvae that were distinct from those in the HK larvae (Fig. 6). A slight variation in expression pattern between the replicates (R1-R3 and R4-R6) of HK larvae was observed. It is  important to note that the adult barnacles were collected from wild natural conditions. Habitat conditions could differ substantially because of factors including origin, age, gender and physiological state. It is difficult to account for uncontrolled sources of variation among specimens collected from natural habitats at the same time in the same area. However, such variability may not have a significant impact as the average individual protein expression was only slightly different among replicates. Furthermore, the average coefficient of variation (CV) among replicate samples from HK was lower (within the standard range for iTRAQ experiments), and not enough to mask the overall difference in the expression patterns of HK samples. Table 2 and Additional file 6: Table  S9 lists the up-regulated proteins and their fold change values. Nine stress-activated proteins were upregulated in the RS larvae relative to the HK larvae (Fig. 7a). The expression of osmoregulatory proteins was also significantly increased in the RS larvae (Fig. 7b). Additional file 7: Table S10 lists some of the down-regulated proteins and their fold change values. In RS larvae 9 proteins involved in cellular detoxification (Fig. 7c) and 11 involved in glycolysis and gluconeogenesis were down-regulated (Fig. 7d).
Correlation between the proteome and transcriptome of RS larvae The number of genes in RS larvae of B. amphitrite that were identified in each functional category, and their corresponding protein expression patterns, are shown in Table 4 and Additional file 9: Table S12. The profiles of the two datasets revealed overlap in the majority of abundant transcripts and differentially expressed proteins. These categories, which included stress response, and development and signaling pathways, showed similar trends of expression at both the transcript and protein levels. We identified 30 stress response proteins from the proteome analysis and 16,367 contigs in the transcriptome; these belonged to various categories of stress. Similarly, the 'developmental' category showed 34 proteins and 24,597 transcripts, while 'signaling pathways' showed 18 proteins and 18,171 transcripts in the respective datasets. Further, the proteins of binding and catalytic activity were also equally represented in the proteome and the transcriptome. Overall, abundant    [16]. The sequencing depth of the present study provides much greater read coverage than the previous studies. Using this in-house transcriptome database we successfully used mass spectrometry to identify 1264 proteins; of these, 170 showed distinct expression patterns between the HK and RS larval populations. We also identified a large number of transcripts related to development, locomotion and signal transduction.

Molecular basis of larval development
The abundant expression of cuticular proteins in both the transcriptome and proteome datasets may reflect the importance of exoskeleton dynamics during development. The barnacle body is covered with exoskeleton, which is composed of carbohydrates and proteins [28]. During molting the larvae partially resorb the cuticle to provide space for newly forming tissues. The larval proteome showed that several structural proteins were differentially expressed. For example, myosins, titin, actin, tubulins, talin-1 and fasciclin-1 were up-regulated in RS larvae (Table 2), whereas settlement pheromone, cement secretion protein 2, retinol dehydrogenase 13 and NMDA receptor protein 1 were down-regulated (Table 3). This is consistent with our previous reports showing differential expression of cytoskeletal proteins during development in polychaetes [29][30][31][32]. The myosins and titins contribute to muscle development and the contraction of striated muscle. In barnacles, striated muscles in antennules and thoracopods help in locomotion and  sensory detection for substratum attachment [33]. Barnacle cyprid-specific protein 2 (BCS-2) is abundantly expressed in cyprids, but decreases following larval attachment [34]. The protein BCL-4 is less abundant in young cyprids, but gradually increases in older cyprids. Apoptosis and protein degradation occur spontaneously during larval development and metamorphosis [35], and are critical in the removal and subsequent degradation of early larval structures, and in cellular and tissue remodeling. In the present study we found several differentially expressed proteins related to apoptosis. Our previous proteomic analyses identified several apoptosis proteins in early stages of larval development in barnacles [19].

Signal transduction pathways
Genes that participate in the MAPK cascade, cGMP, nitric oxide (NO) biosynthesis, calcium and Wnt pathways were identified in the present study. Our previous studies showed that in barnacles these pathways are closely associated with larval settlement and metamorphosis. For example, p38-mediated MAPK signaling has been found to be crucial for larval attachment in barnacles [36,37]. Zhang et al. (2012) found that NO regulates attachment of barnacle larvae by modulating cGMP signaling [38]. It appears that NO signaling contributes to stress-activated responses (in the present case, up-regulation of heat shock proteins), and thereby influences larval survival under unfavorable environmental conditions. Based on our findings, we hypothesize that MAPK, NO, cGMP and calcium signaling are important for larval development and stress tolerance in larvae of B. amphitrite. Furthermore, the proteome data showed up-regulation of six proteins related to G protein signaling or protein kinase. Among these, GTP-binding nuclear protein (RanGTPBP1), GTP-binding protein SAR1B (GTP-BP-SAR1b) and guanine nucleotidebinding protein (GNBPα) belong to G protein signaling, whereas receptor for protein kinase C (RACK2), innexin 2 (INX2) and epidermal growth factor receptor (EGFR15) belong to the protein kinase and calcium receptor-mediated signal transduction cascades. Chen et al. (2014) reported up-regulation of the G protein-coupled receptor in barnacle cyprid larvae [20], and G protein receptors bind to the metamorphic cue, while the protein kinase independent pathway participates in settlement signaling.

Adaptive strategies under extreme marine conditions
Adult barnacles live in various habitats and are subject to differing selection pressures that could have a major influence on the survival strategies of their offspring (in this case nauplii) [39,40]. Temperature and salinity influence larval recruitment and the survival of marine invertebrates [41,42]. In addition, the intertidal zone is highly variable with respect to temperature conditions and among different marine environments [43]. For example, in summer the offshore RS and HK water temperatures average 30.8°C and 28°C, respectively [27,44]. Nauplii may have developed remarkable adaptive mechanisms for survival in the pelagic stage and display a variety of survival strategies. The higher expression levels among stress response genes in RS larvae, and the up-regulation of stress-activated proteins including heat shock proteins (HSPs), chaperonins (CPNs), major egg antigen (MGN), T-complex protein (TCP1), annexin (AnxIXA), cyclophilin (CyPA) and collagen alpha (Col4) suggest the development of adaptive strategies to cope with extreme conditions. This observation is consistent with previous studies showing significant up-regulation of HSPs in barnacle larvae challenged with antifoulants [16,23]. The cellular response of larvae to stress leads to   [45,46]. For instance, in ascidians and gastropods, HSP90 activity regulates the repression of larval metamorphosis by nitric oxide signaling [47]. AnxIXA expression patterns have been shown to change significantly when cells undergo proliferation or differentiation [20]. The differential expression of AnxIXA in RS nauplii and juveniles of B. amphitrite could contribute to antiinflammatory responses. CyPA secreted by cells in response to inflammatory stimuli is known to play an important role in protein folding. We identified a large number of genes involved in osmotic stress tolerance. Furthermore, the RS larval proteome displayed dramatic up-regulation of osmoregulatory proteins including H + K + αATPase, v-type proton ATPase, phosphate transport protein (SLC2C), voltage-dependent anion channel (VDAC2), neuronal acetylcholine receptor (α7-nAChR), carbonic anhydrase (CA) and selenoprotein (SelM) ( Table 2). During barnacle larval development, biomineralization and osmoregulation facilitate larval tolerance to a wide range of osmotic stresses via active ion transport [48]. CA is widely distributed in barnacle tissues, and is involved in the synthesis of calcium carbonate [20] and the molting cycle in crustaceans. In other marine species osmotic stress affects the expression patterns of the proteins ATP6V0A1 and ATPV0B, and Na + −K + −ATPase and V-H + −ATPase are required for ion regulation [49]. The sodium-phosphate symporter SLC2C plays a role in osmoregulation by absorbing phosphate from interstitial fluid. The Ca 2+ transport regulator VDAC plays an important role in regulating the exchange of ions and solute molecules in mitochondria, and α7-nAChR facilitates the opening of ion channels across the plasma membrane. Consequently, we believe that under extreme environmental conditions larvae may be protected by abundant expression of osmoregulatory proteins that maintain cellular homeostasis, and that the large number of genes involved in various homeostasis mechanisms (including for chloride, calcium and sodium ions, and regulation of ion transmembrane and surfactant concentrations) are probably involved in osmoregulation (Fig. 4b).

Metabolic depression
Under extreme environmental conditions marine invertebrates typically show greater metabolic depression by minimizing energy loss [50,51]. It has been estimated that protein translation alone may consume up to 50 % of cellular energy; by suppressing metabolism, energy can be diverted to synthesis of stress tolerance proteins [52]. Metabolic depression may lead to functional cellular modification, post-translational modification of proteins (phosphorylation), and maintenance of homeostasis by ion transport. Consistent with these observations, in RS larvae we found down-regulation of 81 proteins involved in various metabolic processes (Table 3). For instance, glutathione S-transferases (GSTs) are antioxidant isoenzymes that provide cellular defense against oxidative stress and harmful chemicals [53]. Han et al. (2013) reported increased expression of GSTs in barnacle cyprid larvae following exposure to the antifoulant meleagrin [23], suggesting a role in chemical detoxification. GST also regulates apoptosis by inhibiting kinases, thereby affecting downstream signaling pathways. It has been shown in barnacle cyprid larvae that proteins involved in glycolysis and gluconeogenesis are differentially expressed, suggesting roles in maintaining homeostasis [20,23]. Fatty acid binding proteins (FABPs) and fructose 1,6-bisphosphatase (FDPase), which are involved in the synthesis of byproducts of glycolysis and gluconeogenesis, have been identified in barnacle larvae [20]. Membrane proteins play an important role in the transport of solutes and toxic materials across the cell membrane. In particular, mitochondrial inner membrane proteins (MIM and TMIM 23) transport metabolites across the membrane in a highly controlled manner [54]. FABPs act as carrier proteins for fatty acids and other lipophilic substances [55]. The regulation of energetically expensive transport systems appears to be an important strategy used by RS larvae to survive in extreme conditions. The actin binding proteins may regulate cytoskeletal dynamics in developing larvae. It is hypothesized that larval tolerance of stress is regulated by complex stress response mechanism and strategies, and metabolic depression is probably more diverse and complicated than previously assumed. We believe this study is of international significance in advancing marine biology, as most previous studies have focused on response to specific environmental factors in controlled environment; it also highlights the importance of studying the cumulative effects of environmental conditions in natural habitats.

Conclusion
The transcriptome and proteome data generated in this study provide a valuable resource that will enhance molecular studies of crustacean larvae, which are of ecological and economic importance. The large differences found in the protein expression patterns of larval populations from the two contrasting environments were striking. The genes, proteins and signaling pathways identified may play crucial roles in the development of nauplii in the RS environment. For example, the abundant expression of cuticular and muscle proteins in nauplii is critical in the molting cycle, and in muscle development that influences swimming and sensory detection. We also identified genes that participate in several biochemical pathways that are important for larval settlement, metamorphosis and the stress response in barnacles. Specifically, heat shock proteins, chaperonin, major egg antigen, annexin and NO signaling participate in stress-activated responses, and thereby contribute to larval strategies for survival in the RS environment. Proteins involved in osmoregulation, including ATPase, transport proteins, acetylcholine receptor and carbonic anhydrase facilitate larval tolerance to osmotic stress. Overall, the findings of this study contribute significantly to knowledge of larval development and adaptation in extreme conditions, and the settlement biology of crustaceans.

Environmental conditions
Marine environmental data were collected from each sampling location. Measurements were carried out using a YSI Professional Pro Plus Multiparameter meter (YSI, Ohio, USA), which was calibrated prior to use. The probe was immersed in the water at the designated locations, and measurements were recorded after 2-3 min; it was rinsed with fresh water between measurements and locations.  Table S13).

Barnacle collection
Barnacles were collected from the intertidal zone between August 2012 and November 2012. Adult B. amphitrite barnacles attached to hard rocks were collected from the intertidal rocky shore of the King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia (27°18′168″N, 35°05′661″ E). Approximately 100 collected adults were washed to remove attached debris and sediment, and were kept in a 10 L tank containing aged filtered sea water. The tank was covered with black plastic sheet, but had a small opening enabling light to penetrate, as a means of attracting newly released larvae. Newly hatched nauplii (hereafter, newly released RS larvae) were collected at 30-min intervals using a pipette until sufficient larvae were obtained for subsequent studies. Excess water was removed and approximately 3-4 × 10 3 larvae derived from independent replicates were separated into two groups, one of which was used for transcriptome analysis and the other for proteome analysis (Additional file 11: Figure S1).
Approximately 100 individuals of adult B. amphitrite barnacles were collected from concrete columns at Pak Sha Wan, HK (22°21′45″N, 114°15′35″E), and placed in a 10 L tank containing filtered seawater (FSW; 0.22 μm) to induce the release of larvae. Approximately 3-4 × 10 3 newly hatched nauplii (hereafter, newly released HK larvae) were collected as described above, and transferred overnight on dry ice to the King Abdullah University of Science and Technology. No significant size difference in the adults from the two populations was observed. On arrival the larvae were thawed, washed in seawater, and used for proteome analysis.
For the RS larval samples the total RNA was extracted from 600 to 700 larvae using TRIzol reagent (Invitrogen), following the manufacturer's instructions. TURBO DNA-free treatment was performed using RNeasy spin columns (Qiagen). The RNA was purified and eluted in 14 μl of MilliQ water. The purity was checked using a NanoDrop ND-1000 UV-vis spectrophotometer (Nano-Drop Technologies). The integrity of the extracted RNA was then checked using an Agilent 2100 Bioanalyzer (Agilent Technologies), and confirmed on a 1 % agarose gel.
Library preparation and Illumina sequencing cDNA library preparation was performed according to the Illumina TruSeq standard RNA Seq library preparation protocol, and sequencing was performed using an Illumina HiSeq 2000 genome sequencer. Briefly, the quantity of total RNA was measured using a Qubit® 2.0 fluorometer (Invitrogen). rRNA was removed using the Ribo-Zero Magnetic Gold Kit (Epicenter, Illumina). The RNA was subjected to thermal fragmentation, and first strand cDNA was synthesized using Super-Script III reverse transcriptase (Invitrogen). The cDNA was converted into double stranded DNA using the Second Strand Making Master Mix (Illumina). After dA-tailing, ligation of adaptors and purification, library amplification (15 cycles) was performed using a thermal cycler (Applied Biosystems). The libraries were purified using AMPure XP beads (Beckman Coulter, Inc.). Library dilution and pooling was carried out as described in the Illumina standard protocol. A quantity of 7.5 picomoles of DNA was used for cluster generation (CBOT Illumina) and sequencing.

Assembly and annotation
Low quality reads (Q-score < 20) and adapters were trimmed using java scripts designed in-house. Reads were assembled using ABySS v1.3.4 [56] with setting parameters of every odd k-mer from 45 to 85, and consolidated using Trans-ABySS v1.4.4 [57]. Contig redundancy was reduced using CD-HIT-EST with the threshold sequence identity set to 0.99, to discard contigs with < 10 bp mismatches per 1 kb [58]. To obtain coding amino acid sequences we used GETORF to translate contigs from start to stop codons for all six possible reading frames [59]. Two approaches were used for functional annotation of contigs. First, homologous proteins from other species were searched using blastp against a non-redundant (nr) database (minimum bit score threshold 50; match length 20 amino acids). Second, the PFAM domain of the proteins was searched using Interproscan v5.4 [60]. Gene ontology (GO) and Reactome pathway information from homologous genes was transferred to barnacle contigs using a transcript designed in-house.

Larval proteome extraction, digestion and iTRAQ labeling
The HK and RS larvae were suspended in 8 M urea and protease inhibitor (Roche Diagnostics, Germany) and homogenized (Wheaton homogenizer, USA). The homogenate was sonicated at 4 W using five × 1 s bursts (Q Sonica, LLC, USA), and the protein concentration was measured using a 2-D Quant kit (GE Healthcare, United Kingdom). Protein (100 μg) from each sample was reduced, alkylated, and diluted by a factor of 8 using 50 mM iTRAQ dissolution buffer as previously described [61]. The protein was digested using a trypsinprotein ratio of 1:50 for 14-16 h at 37°C (Promega, USA). The digest was desalted using Sep-Pak C18 Vac cartridges (Water Corporation, USA), and labeled using the iTRAQ Reagents Multiplex (4plex) Kit (Applied Biosystems, USA), according to the manufacturer's protocol. Briefly, dried peptides were reconstituted in 30 μL of resolubilization buffer, and 70 μL of ethanol was added to the iTRAQ reagents; the reagents 114 and 115 were labeled to HK larval peptides, and reagents 116 and 117 were labeled to the RS larval peptides. Labeling was carried out at room temperature for 60 min, and the labeled peptides were multiplexed and dried prior to fractionation.

Peptide fractionation coupled with mass spectrometry
The iTRAQ-labeled peptides were suspended in 85 μL strong cation exchange chromatography (SCX) buffer A, and centrifuged at 10,000 rpm for 5 min at room temperature. The supernatant containing peptides was transferred to liquid chromatography (LC) sample vials and fractionated using an Accela 1250 LC system (Thermo Scientific, USA). The SCX-fractionated peptides were desalted using Sep-Pak C18 Vac cartridges and resuspended in 20 μL of LC sample buffer (97 % H 2 0, 3 % ACN, 0.1 % formic acid), centrifuged at 10,000 rpm for 5 min, and then transferred to LC-MS sample vials. A total of 15 SCX fractions were run in a LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Germany) coupled to a Proxean Easy-nLC liquid chromatography system (Bruker, Denmark), as described previously [61].

Protein identification
Raw MS data were processed following a previously described procedure [62]. Briefly, higher-energy collisional dissociation (HCD) and collision-induced dissociation (CID) spectra were extracted independently using Proteome Discoverer 1.2 software (Thermo Scientific), and processed using a script designed in-house. Mascot generic format (MGF) files were submitted to MASCOT v2.2 (Matrix Sciences Ltd, United Kingdom) for searching against a newly released RS barnacle larval protein dataset (92,117 target sequence entries that matched to 43,921 unique GI numbers), developed in-house from transcriptome data. The mass tolerance for the peptide was set to 10 ppm, and the MS/MS fragment ion tolerance was 0.5 Da. A maximum of one missed cleavage was allowed. Variable modifications were set to 4-plex iTRAQ and oxidation was set to methionine (M). The fixed modifications were set to methylethanethiosulfonate at cysteine and lysine, and 4-plex iTRAQ at Nterminal. The MASCOT files (.dat files) were analyzed using Scaffold v4.1.1 (Proteome Software Inc. USA) software for validation and quantitation of peptide and protein identifications. The peptide and protein identification threshold was set at 95 % using the Peptide and Protein Prophet algorithm with Scaffold delta-mass correction [63,64]. Using these criteria the false positive rates for peptide and protein were 1 % and 0.1 %, respectively.

Protein quantitation
iTRAQ label-based quantitation of the identified proteins was performed using the Scaffold Q+ algorithm. The intensities of all labeled peptides were normalized across all runs. Individual quantitative data acquired in each run were normalized using the i-Tracker algorithm, as described previously [65]. Peptide intensity was normalized within the assigned protein. The reference channel (e.g. 114) was normalized to produce a 1:1 fold change, and the iTRAQ ratios were then transformed to a log scale. P values were calculated using a paired t-test. All normalization calculations were performed as described previously [61].

Statistical analysis
The sources of variation between biological and technical replicates of HK and RS samples were evaluated using unsupervised multivariate principal component analysis (PCA), using the Multiple Array Viewer (MeV) data analysis and visualization tool [66]. Log2transformed fold changes from six replicates were used for the analysis. Sample selection was set to Cluster, and