Comprehensive and Quantitative Proteomic Analysis of Metamorphosis-Related Proteins in the Veined Rapa Whelk, Rapana venosa

Larval metamorphosis of the veined rapa whelk (Rapana venosa) is a pelagic to benthic transition that involves considerable structural and physiological changes. Because metamorphosis plays a pivotal role in R. venosa commercial breeding and natural populations, the endogenous proteins that drive this transition attract considerable interest. This study is the first to perform a comprehensive and quantitative proteomic analysis related to metamorphosis in a marine gastropod. We analyzed the proteomes of competent R. venosa larvae and post-larvae, resulting in the identification of 5312 proteins, including 470 that were downregulated and 668 that were upregulated after metamorphosis. The differentially expressed proteins reflected multiple processes involved in metamorphosis, including cytoskeleton and cell adhesion, ingestion and digestion, stress response and immunity, as well as specific tissue development. Our data improve understanding of the physiological traits controlling R. venosa metamorphosis and provide a solid basis for further study.


Introduction
The veined rapa whelk (Rapana venosa) is an economically important sea snail in China, and since 1992, there has been interest in its commercial aquaculture [1]. However, sea-ranching efforts have been hampered by difficulties cultivating larvae during the settlement and metamorphosis stages. In countries that do not consume R. venosa, such as the United States, Argentina, and France, this predatory species has become an invasive pest due to unintended worldwide transport and severely disrupts the survival of native bivalves [2][3][4][5][6]. Because R. venosa population dynamics and spatial expansion are dominated by recruitment and survival rate during metamorphosis, which is a vital process in the species' biphasic life cycle, understanding the mechanisms behind this process is necessary for both successful aquaculture and invasion control. Moreover, the metamorphosis of R. venosa is unusual compared with other lifelong phytophagous gastropods for exhibiting considerable developmental specificity; the planktonic, pelagic larvae go from filter-feeding on microalgae to carnivorous juveniles that prey on bivalves [7]. This transition occurs rapidly, despite fundamental changes in morphology including velum degeneration and reabsorption, foot reorientation and elongation, as well as secondary-shell growth [7]. Thus, clarifying R. venosa metamorphosis is also of theoretical interest to gastropod researchers.
However, information about R. venosa metamorphosis is relatively scarce. A previous study had documented the morphological changes that occur during this process [7]. Additionally, CaCl 2 and acetylcholine chloride were found to be effective and low-toxicity inducers of metamorphosis in R. venosa pelagic larvae [8], suggesting these compounds might be suitable for applying to its artificial seeding. Finally, a comprehensive transcriptomic profile has been constructed from R. venosa planktonic larvae and post-larvae [9], which paves the way for studies on metamorphosis-related gene activity. However, because complex gene regulation occurs during post-transcription and post-translation [10,11], proteomic data are required to provide more concrete support for conclusions based on transcriptome data. Indeed, proteomic analysis has been successfully applied to identify a number of metamorphosis-related proteins in marine-invertebrates, specifically in bryozoans [12], polychaetes [13,14], and barnacles [12,15]. To our knowledge, no proteomic study has been conducted to investigate gastropod metamorphosis.
Although two-dimensional electrophoresis (2DE) is the most common proteomic approach, the method lacks the sensitivity to identify low-abundance proteins or those not amenable to gels [16]. Moreover, 2DE's accuracy is potentially compromised by the phenomenon of protein co-migration [17]. The recently developed, high-throughput isobaric tag for relative and absolute quantitation (iTRAQ) has therefore become increasingly popular. This method labels peptides with isobaric (same-mass) reagents consisting of reporter ions and their equalizing balance groups. During mass spectrometry (e.g., collision-induced dissociation (CID)), the reporter ions are then separated from the labelled peptides, allowing for determination of ion intensity and thus peptide quantity. Therefore, iTRAQ differs from other quantitative proteomics technologies, which tend to measure precursor (pre-fragmentation) ion intensities. The difference allows for greater accuracy and reliability [18]. In this current study, we chose iTRAQ to assess proteomic changes during metamorphosis via a comparative proteomic analysis on competent larvae and juveniles of R. venosa. We were able to identify and annotate over 5000 proteins through searching the R. venosa transcriptome with protein sequences [9]; 1138 of the identified proteins were differentially expressed, during metamorphosis, suggesting that they are responsible for the process. Our results showed that these differentially expressed proteins function in diverse biological processes, including cytoskeleton and cell adhesion, ingestion and digestion, stress response and immunity, as well as specific tissue development. These findings provide a proteomic overview of gastropod metamorphosis and facilitate future research on protein function during the transitions of a biphasic life cycle.

General Characterization of Proteomic Data
Raw data have been deposited to the ProteomeXchange Database (accession number: PXD004119). As shown in Table 1, of the 224,473 detected spectra, 46,485 were considered unique. Moreover, 5321 proteins were identified. Figure 1 displays the overall changes to protein abundance before and after metamorphosis. More detailed information on these 5321 proteins is available in Table S1, while variation in expression during metamorphosis is shown in Table S2: 470 proteins were upregulated and 668 were downregulated after metamorphic transition (Table S2). Homologous sequence analysis of these differentially expressed proteins (DEPs) revealed four functional groups of interest (Table 2): cytoskeleton and cell adhesion, ingestion and digestion, stress response and immunity, as well as specific tissue development.

Functional Analysis of DEPs with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
Under GO analysis, significant enrichment (p < 0.05) was found for 77, 27, and 63 categories in the biological process (BP), cellular component (CC), and molecular function (MF) domains, respectively (Table S3). The most enriched GO terms were metabolic, cellular, and single-organism processes in BPs; cell and cell part in CCs; as well as binding and catalytic activity secondary items in MFs (Figure 2).

Functional Analysis of DEPs with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
Under GO analysis, significant enrichment (p < 0.05) was found for 77, 27, and 63 categories in the biological process (BP), cellular component (CC), and molecular function (MF) domains, respectively (Table S3). The most enriched GO terms were metabolic, cellular, and single-organism processes in BPs; cell and cell part in CCs; as well as binding and catalytic activity secondary items in MFs ( Figure 2).

Functional Analysis of DEPs with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
Under GO analysis, significant enrichment (p < 0.05) was found for 77, 27, and 63 categories in the biological process (BP), cellular component (CC), and molecular function (MF) domains, respectively (Table S3). The most enriched GO terms were metabolic, cellular, and single-organism processes in BPs; cell and cell part in CCs; as well as binding and catalytic activity secondary items in MFs ( Figure 2). Of the 38 significantly enriched pathways under KEGG analysis (p < 0.05; Table S4), seven were reliably enriched after adjustment (q < 0.05; Table 3). The high representation of phototransduction, pentose and glucuronate, olfactory transduction, and salivary secretion pathways suggest changes to ingestion and digestion characteristics during metamorphosis. Additionally, enrichment in Of the 38 significantly enriched pathways under KEGG analysis (p < 0.05; Table S4), seven were reliably enriched after adjustment (q < 0.05; Table 3). The high representation of phototransduction, pentose and glucuronate, olfactory transduction, and salivary secretion pathways suggest changes to ingestion and digestion characteristics during metamorphosis. Additionally, enrichment in glycerolipid metabolism and galactose metabolism pathways illustrate differing energy strategies between competent larvae and post-larvae.

Association Analysis of Transcriptome and Proteome Data
We performed a direct comparison of transcriptome and proteome abundance during metamorphosis. Concordance tests revealed a significant relationship between mRNA and protein ratios (Pearson's correlation, r = 0.3699; Figure 3). We observed 458 concordant dots, representing a correspondence of protein abundance with transcript accumulation (red dots in Figure 3). We also found 282 green dots and 592 blue dots, respectively, indicating differential expression only on the transcript or the protein levels. Detailed quantitation and annotation on the points in Figure 3 are provided in Table S5. glycerolipid metabolism and galactose metabolism pathways illustrate differing energy strategies between competent larvae and post-larvae.

Association Analysis of Transcriptome and Proteome Data
We performed a direct comparison of transcriptome and proteome abundance during metamorphosis. Concordance tests revealed a significant relationship between mRNA and protein ratios (Pearson's correlation, r = 0.3699; Figure 3). We observed 458 concordant dots, representing a correspondence of protein abundance with transcript accumulation (red dots in Figure 3). We also found 282 green dots and 592 blue dots, respectively, indicating differential expression only on the transcript or the protein levels. Detailed quantitation and annotation on the points in Figure 3 are provided in Table S5.

Discussion
In this study, we performed a proteomic analysis to identify DEPs before and after R. venosa metamorphosis. Based on the reference transcriptome, we identified 470 upregulated proteins and 668 downregulated proteins. These DEPs were generally associated with cytoskeleton and cell adhesion, ingestion and digestion, immunity and stress response, transcription and translation, specific tissue development, and signal transduction. Additionally, their differential expression patterns reflect life-stage transitions in R. venosa (Table 2). We discuss the implications of our results in the following sections.

Cytoskeleton and Cell Adhesion
The intracellular cytoskeleton, transmembrane cell-adhesion components, and extracellular matrices (ECMs) comprise a complex "skeleton" network, which is critical for cell motility processes, including proliferation, differentiation, migration, and apoptosis. In this study, active cell motility during metamorphosis is indicated by the abundance of proteins involved in cytoskeleton, cell adhesion, and ECMs.
Tubulins (tubulin α-1 chain, tubulin α-2 chain, tubulin β-2 chain, and tubulin β-4B chain) were highly expressed in larvae but declined in post-larvae. As components of microtubules, alpha and beta tubulins function in essential cellular processes, including cell division, proliferation, and migration [19]. Any temporal variation in tubulin expression is likely related to various physiological functions and post-translational modifications [20,21]. Thus, the expression patterns that we observed are consistent with the suggestion that protein degradation and apoptosis during metamorphosis mediate the loss of larval organs, as well as the morphogenesis of juvenile characteristics [22,23]. Furthermore, our results conformed with studies in marine invertebrates (e.g., the spionid polychaete Pseudopolydora vexillosa [24] and polychaete Hydroides elegans [13]) that demonstrated a decline of tubulin isoforms during metamorphosis.
Proteins associated with ECMs were also differentially expressed. Specifically, we observed downregulation in collagen α-1 (XV, XXI, and XXII chain), collagen α-6 (VI chain), and matrix metalloproteinase-19. The ECM is the cell base and participates in tissue remodeling, as well as cell migration and differentiation; convincing evidence exists to show that ECMs are remodeled during metamorphic transition [25,26], and, in fact, the process is considered essential in the metamorphosis of amphibians [26,27], insects [28], and mollusks [29]. Thus, the observed expression patterns suggest that ECM remodeling-specifically involving the identified proteins-functions in R. venosa metamorphosis. Although this hypothesis requires further validation for our study species, we note that collagenase (a matrix-metalloprotease) was first discovered in the tail of a tadpole undergoing metamorphosis [30]. Additionally, matrix metalloprotease was highly expressed during the metamorphosis of the lepidopteran Galleria mellonella, causing collagen degradation [31].

Ingestion and Digestion
Morphological and functional changes in the digestive system clearly play a vital role in the metamorphic transition of R. venosa from a diet of microalgae to one of bivalve mollusks [7]. It follows that proteins associated with food intake and digestion will be differentially expressed between the larval and post-larval stages. Indeed, we found that post-metamorphosis, carnivorous digestive enzymes clearly increased, whereas phytophagous digestive enzymes were downregulated. Our study provides novel molecular data on the dietary shift that occurs with metamorphic transition.
In larval R. venosa, we detected several enzymes involved in the breakdown of cellulose and hemi-cellulose, both plant cell-wall components. Specifically, we observed two important cellulase components, endoglucanase and exoglucanase, as well as endo-1,4-β-xylanase, important in the hydrolysis of hemicellulose. Next, we also observed the presence of β-galactosidase, a key enzyme in the hydrolysis of lactose into galactose and glucose. Together, these data indicated that larval whelks were able to completely digest and absorb microalgae. High levels of cellulases have been reported in the pre-competent and competent larvae of the spotted babylon snail Babylonia areolata, which also has a pre-metamorphosis diet of microalgae [32], suggesting that the two species may have similar digestive mechanisms.
In R. venosa post-larvae, we observed higher levels of proteolytic enzymes, illustrating the capacity to exploit varied protein diets post-metamorphosis. For example, serine proteases (trypsin and chymotrypsin), as well as zinc carboxypeptidase, are major proteolytic enzymes in the gastropod digestive glands and were all highly expressed. Additionally, we observed an upregulation of pancreatic triacylglycerol lipase in post-larvae. Through hydrolysis, lipases prepare fatty acids for absorption through membranes [33]. Our results are corroborated by previous findings of high lipase expression in B. areolata juveniles [32]. Taken together, we suggest that cellulase downregulation and protease/lipase upregulation are primarily responsible for the transition from herbivores to carnivores in R. venosa with biphasic life history.
Unexpectedly, we found high expression of conotoxin and cysteine-rich venom protein in the post-larvae. The former is a neurotoxic peptide that was first isolated from the venom of the predatory marine cone snail (Conus spp.) [34]. The latter has also been found in a particular species of cone snail, Conus textile, where it exhibits protease activity and functions in pro-conotoxin processing of C. textile venom [35]. Our results suggest that R. venosa may possess predation mechanisms homologous to Conus. As little information is available regarding the composition and toxicity of R. venosa venom, the presence of conotoxin observed here warrants further research.
In summary, the diverse suite of proteins associated with ingestion and digestion illustrates the capacity of R. venosa to exploit different diets that suit the shifting nutritional requirements in a biphasic life cycle.

Stress Response and Immunity
Proteins involved in stress response and immunity tend to be upregulated during metamorphosis [36]. In the present study, we found that anti-oxidant enzymes, such as thioredoxin-T and peroxiredoxin-2, were highly expressed in the competent larval stage. Similarly, significant upregulation of peroxiredoxin has been documented in Crassostrea gigas post-metamorphosis [29]. These patterns suggest that competent larvae may experience considerable oxidative stress from reactive oxygen species (ROS) [29]. Indeed, amphibian studies have shown that when endogenous thyroid hormone induces metamorphosis, it also enhances mitochondrial respiration, which leads to higher ROS content [37,38]. Similar mechanisms may be at work in R. venosa, and the observed anti-oxidant enzymes are likely essential for protection against ROS-induced cell damage and maintenance of cell redox homeostasis during the metamorphosis.
We also noticed that R. venosa hemocyanin (RvH) A-type and RvH G-type were significantly upregulated after metamorphosis. Hemocyanin was first identified in the snail Helix pomatia; the protein has two copper atoms that reversibly bind with oxygen and acts as an oxygen transport molecule similar to hemoglobin. Under cold environments with low oxygen pressure, hemocyanin is more efficient at oxygen transportation than its vertebrate counterpart [39]. However, hemocyanin also plays important roles in innate immunity, exhibiting antiviral, antimicrobial, and antitumor activities [40,41]. Further evidence supporting this role in immune function includes the identification of four novel proline-rich peptides from RvH that exhibit antimicrobial activities against Gram-positive Klebsiella pneumonia and Gram-negative Staphylococcus aureus [42]. Moreover, the structural subunits RvH-1 and RvH2 exert strong antiviral effects upon the Herpes simplex virus [43,44]. Thus, two complementary levels of explanation could account for abundant RvH expression in juvenile R. venosa: on the evolutionary level, it is an adaptation to hypoxia stress at the benthic life stage, and on the developmental level, it reflects immune-system maturation post-metamorphosis. In support of the latter concept, proteins such as α-2macroglobulin and myeloperoxidase were also elevated in post-larvae. α-2-macroglobulins are selective protease inhibitors and major components of the eukaryotic innate immune system [45], while myeloperoxidase is highly expressed in neutrophil granulocytes, where it produces antimicrobial hypohalous acids [46].

Specific Tissue Development
Tissue-specific or tissue-preferential DEPs likely reflect physiological changes in those tissues [29]. For example, fluctuations in tropomyosin and myosin abundance are closely associated with muscle development during the metamorphosis of red abalone Haliotis rufescens [47,48]. Here, we demonstrated that larvae and post-larvae exhibit differential expression of neuron-and muscle-specific proteins, including myosin heavy chain, myosin light chain, and neuroglian proteins. All of these proteins are closely involved with transitions in nervous and muscular systems during molluscan metamorphosis [36,48].
As described earlier (see Section 3.1), tubulins were downregulated after metamorphosis. These proteins are cilia-specific, along with tektin, dynein heavy chain, and dynein beta chain, all of which experienced downregulation. The decline of proteins that comprise core cilial structure and function in cilia movement accords with post-metamorphic degradation of the velum, a conspicuous, ciliated organ in larvae used for swimming and filter-feeding.

Larvae Culture and Sample Collection
Egg capsules of Rapana venosa were obtained from rocks in Laizhou Bay (37˝11 1 4.78 2 N, 119˝41 1 3.75 2 E), Laizhou, China. Larvae were cultivated at Blue Ocean Co. Limited (Laizhou, China) following previously published methods [7]: pelagic larvae were cultured in 2.5 mˆ2.5 mˆ1.5 m tanks with a density range of 0.3-0.05 ind/mL, depending on developmental stage. Diets were a mixture of microalgae Platymonas subcordiformis, Isochrysis galbana, and Chlorella vulgaris; larvae were fed 13.0ˆ10 4 cell/mL daily. Seawater used for culturing was filtered with sand and radiosterilized with UV light. Water temperature was maintained below 25˘1˝C. Larvae samples from four spiral-whorl stages (competent larva) and post-larval stages were collected and examined under a microscope to guarantee developmental synchronies. Samples were immediately washed with dH 2 O, snap frozen in liquid nitrogen, stored at´80˝C till use.

Protein Extraction, Digestion, and iTRAQ Labelling
Three biological replicates (each containing approximately 500 mg larvae) were prepared for the iTRAQ analysis. Total proteins were extracted using the cold acetone method. Samples were ground to powder in liquid nitrogen before the addition of 2 mM EDTA and 1 mM PMSF, then dissolved in lysis buffer. After 5 min, DTT (10 mM) was added to the samples, which were centrifuged at 4˝C and 25,000ˆg for 20 min. All subsequent centrifugation steps described in this section occurred at 4˝C and 25,000ˆg. The precipitate was then discarded and the supernatant was mixed with 10 mM DTT in 5ˆvolume of cold acetone, followed by incubation at´20˝C for 12 h. After a second round of centrifugation for 20 min, the supernatant was discarded. Pellets were washed in 1.5 mL cold acetone (containing 10 mM DTT), then centrifuged a third time for 15 min, to discard the supernatant. This final step was repeated three times. The precipitate was then air-dried and resuspended in 1 mL extraction buffer (10 mM DTT, 4% (w/v) CHAPS, 30 mM HEPES, 8 M urea, 1 mM PMSF and 2 mM EDTA), sonicated for 10 min, and centrifuged for 15 min. The resulting supernatant was transferred to a new tube, mixed with 10 mM DDT, and incubated at 56˝C for 1 h. The solution was incubated in a dark room for another hour after the addition of iodacetamide (55 mM), then precipitated in cold acetone at´20˝C overnight. Finally, the precipitate was centrifuged for 15 min, air-dried, and dissolved in 1 mL extraction buffer under ultrasound. Protein quality and concentrations were examined with SDS-PAGE and the 2-D Quant Kit (General Electric Company, Fairfield, CT, USA), respectively.
Protein digestion was performed with Trypsin Gold (Promega, Madison, WI, USA) for 16 h at 37˝C, and peptides were dried in a centrifugal vacuum concentrator. Competent-larvae samples were labeled with iTRAQ tags 113, 114, and 115, whereas post-larvae samples were labeled with tags 118, 119, and 121, following manufacturer protocol in the iTRAQ 8-plex labelling kit (Applied Biosystems, Foster City, CA, USA).

Protein Identification and Quantification
Raw data from LC-MS/MS were transformed into MGF files with Proteome Discovery 1.2 (Thermo, Pittsburgh, PA, USA). In the Mascot search engine (version 2.3.02, Matrix, Science, London, UK), proteins were identified using the R. venosa reference transcriptome [9]. Mascot search results were then normalized and quantified. Proteins with fold changes significantly (p < 0.05) >1.2 or <0.83 were considered differentially expressed [49].

Enrichment of GO and KEGG Pathways
We searched against the GO and KEGG databases to classify and identify differentially expressed proteins [50,51]. Significant pathway enrichment was examined with the hypergeometric test, and significance was set at p < 0.05.

Correlation Analysis of Transcriptomic and Proteomic Data
Previously, we had constructed an RNA-seq library of competent larvae and post-larvae (raw data available in NCBI GEO, accession number GSE70548). To investigate the concordance between transcript and protein levels, we calculated the Pearson's correlation for these data and created scatterplots with the expression ratios of competent larvae versus post-larvae.

Conclusions
Using iTRAQ, we constructed a comprehensive and quantitative proteomic profile of R. venosa larvae and post-larvae. To our knowledge, this work is the first proteomic study focused on gastropod metamorphosis. We identified over a thousand differentially expressed proteins that reflected physiological processes occurring in metamorphosis, including changes to cytoskeleton and cell adhesion, ingestion and digestion, stress response and immunity, as well as tissue development.
Our data contributed to a better understanding of the regulatory mechanisms underlying R. venosa development through identifying major participating proteins. Therefore, this study should provide a sound basis for future studies aiming to investigate specific metamorphosis-related proteins in greater depth.