Proteomic Changes between Male and Female Worms of the Polychaetous Annelid Neanthes arenaceodentata before and after Spawning

The Neanthes acuminata species complex (Polychaeta) are cosmopolitan in distribution. Neanthesarenaceodentata , Southern California member of the N . acuminata complex, has been widely used as toxicological test animal in the marine environment. Method of reproduction is unique in this polychaete complex. Same sexes fight and opposite sexes lie side by side until egg laying. Females lose about 75% of their weight and die after laying eggs. The male, capable of reproducing up to nine times, fertilizes the eggs and incubates the embryos for 3-4 weeks. The objective of this study was to determine if there is any set of proteins that influences this unique pattern of reproduction. Gel-based two-dimensional electrophoresis (2-DE) and gel-free quantitative proteomics methods were used to identify differential protein expression patterns before and after spawning in both male and female N . arenaceodentata . Males showed a higher degree of similarity in protein expression patterns but females showed large changes in phosphoproteme before and after spawning. There was a decrease (about 70%) in the number of detected phosphoproteins in spent females. The proteins involved in muscular development, cell signaling, structure and integrity, and translation were differentially expressed. This study provides proteomic insights of the male and female worms that may serve as a foundation for better understanding of unusual reproductive patterns in polychaete worms.


Introduction
Polychaete species are widely distributed in the marine environment and inhabit estuaries to deep-hydrothermal vents [1]. Members of the polychaete Neanthes acuminata species complex are cosmopolitan in distribution [2,3]. Populations from the east coast of North America are referred to as N. acuminata; species from Europe are known as N. caudata; southern California as N. arenaceodentata; and Asia as N. crigognatha. All are morphologically identical and have the same reproductive characteristics [3]. The laboratory populations of N. arenaceodentata have been used in many studies: toxicity [4,5], reproductive behavior [3], vitellogensis [6], feeding rate metabolism [7] and nervous system architecture [8]. The reproductive behavior is unusual and unique in the genus [2]. Same sexes fight and opposite sexes lie side by side within a mucoid tube until female lays her eggs. The female dies after laying eggs, and the male incubates the fertilized eggs for three to four weeks. During embryonic development the male undulates his body within his tube which insures clean water and expelling metabolic wastes. Males will fight off both sexes during the time period of incubation. About 30% of the time the male will eat the embryos or developing larvae which is probably the result of disturbance or insufficient food. The body muscles of the female during the process of gametogenesis are reabsorbed and supply the material for the large yolky eggs (500-600 µm in diameter). Females who have laid eggs are referred to as spent females. Her locomotive ability is reduced after egg laying; her digestive tract is complete but she is unable to protrude her proboscis to feed because of reduced musculature and may either leave the tube or be eaten by the male [2]. Males are capable of reproducing up to 9 times [4]. Given the uniqueness of N. arenaceodentata's behavior and reproduction, the objective of this research is to characterize the proteome and protein modification (phosphorylation) dynamics to determine why the female can only reproduce once and the male many times.
In recent years, proteomic analyses have been used to compare proteome patterns in polychaetes and other worm species. Previously we reported changes in patterns of proteins and phosphoproteins during early larval development of N. arenaceodentata [9]. OMIC analysis also provided information on genes/proteins and molecular pathways during larval metamorphosis of the polychaete Pseudopolydora vexillosa [10]. Proteomic analysis revealed invasion and habitat adaptation success in two closely related polychaetes Marenzelleria neglecta and Marenzelleria viridis [11]. Transcriptome and proteome analysis provided insights in understanding metal resistance in Ophelina sp. [5]. In other species, proteomic techniques resulted in the identification of proteins during reproductive development. For example, identification of differentially expressed proteins provided insights into the development in the male and female trematode Schistosoma japonicum [12]. Tissue-specific proteins were identified in the male reproductive system of Drosophila melanogaster [13] and stage-and gender-specific proteins in the nematode Brugia malayi [14].
The objective of this study is to identify signature proteins during the reproductive period of male and female N. arenaceodentata. Gel-based analysis indicated that the proteome abundance was highly conserved in male worms but significantly different in spent female worms. Furthermore, owing to limitations of two-dimensional gel electrophoresis (2-DE), gel-free proteomics was used to determine quantitative proteomic differences between male and females.

Specimen culture
Living Neanthes arenaceodentata were collected from Los Angeles Harbor, USA in 1964. A population was established then and has undergone over 200 generations in the laboratory. All descendants are derived from the original collection of six animals. The following life cycle stages were used in this study: mature virgin males (MM), males after first spawning (MAS), females with maturing ova (MF), and spent females (SF) (Figure 1). A minimum of 10 worms of each stage were frozen and shipped in dry ice by overnight express to Hong Kong. Worms were thawed and washed in seawater to remove debris.

2-DE and image analysis
The protein extraction and 2-DE was performed as described by Chandramouli et al. [11]. Worms were transferred to lysis buffer (7 M urea, 2 M thiourea, 4% [3-[(3 cholamidopropyl) dimethylammonio]-1-propanesulfonate] (CHAPS), 40 mM dithiothreitol (DTT) and protease inhibitors (Roche Applied Science, Mannheim, Germany) and then sonicated on ice using 10 sec blasts of 15% amplitude. The homogenate was centrifuged at 13,000 rpm for 15 min and the supernatant protein was purified using a 2-DE cleanup kit (Bio-Rad, Hercules, CA, USA). The protein pellets were solubilized in lysis buffer and quantified using an RC-dC kit (Bio-Rad, Hercules, CA, USA). Three hundred µg of each sample were rehydrated in a buffer (7M of urea, 2M of thiourea, 4% CHAPS, 40mM of DTT, 0.5% pI 4-7 ampholyte, and 1% bromophenol blue) on 17 cm immobilized pH gradient (IPG) strips (pH 4-7) for 14 hr. Isoelectrical focusing (IEF) was performed at 250 V for 20 min, followed by a gradient from 1,000 to 8,500V to reach a total of 60,000Vh on Protean IEF Cell (Bio-Rad, Hercules, CA, USA). The 2-DE gels were fixed for 1 hr in a mixture of 40% methanol and 10% acetic acid and stained for phosphoproteins using ProQ Diamond dye (Invitrogen, Eugene, OR, USA) for 3 hr (1 hr intervals of three times). The gels were washed and scanned using a Typhoon trio imager (GE Healthcare, Piscataway, NJ, USA). These gels were then stained for total proteins using Sypro Ruby dye (Invitrogen, Eugene, OR, USA) and scanned using the Typhoon trio imager. Quantitative and qualitative data for protein spots of three biological replicate gels were obtained by PDQuest software (Bio-Rad, Hercules, CA, USA) as described by Thiyagarajan et al. [15]. Spot detection in each gel was verified by manual inspection of spots to obtain a similar pattern of spots for analysis.

Dephosphorylating phosphoprotein spots by phosphatase treatment
Protein was extracted from mature worms and phosphatase treated as described in Chandramouli et al. [11]. Two phosphatase (λ-PPase) treated (New England Bio labs, Ipswich, MA, USA) and untreated protein samples were incubated overnight at 30 0 C. The samples were purified using 2-DE cleanup kit and and then analyzed using 2-DE method as described above. The gels were stained for phosphoproteins using ProQ Diamond dye and scanned using a Typhoon trio imager.

Mass spectrometry
The differentially expressed or abundant stage-specific protein spots were excised and digested in 20 µl of 12.5 ng/ml trypsin (Promega, Madison, WI, USA) for 16 hr at 37 o C. The peptides were extracted, dried, reconstituted in 15 µl of 0.1% formic acid, and purified using C18 ZipTips (Millipore, Bellirica, MA, USA). The purified dried peptides were subjected to nanoflow UPLC (nanoAcquity, Waters, Milford, MA, USA) coupled with an ESI-hybrid Q-TOF (Premier, Waters, Milford, MA, USA) tandem mass spectrometer as described in Zhang et al. [16]. The instrument was set to positive ion data-dependent acquisition mode with smass range of 300-1600 m/z. Three abundant peptides with +2 to +4 charge were selected for MS/MS analysis. Protein identification was achieved using the in-house built transcriptome database of the polychaete Hydroides elegans.

Quantitative proteomics: Protein extraction, peptides labeling and fractionation
Sample preparation for quantitative proteomics was carried as described by Chandramouli et al. [10] with modifications. The proteins from MM and MF worms were solubilized in 8M urea and quantified using an RC-dC kit (Bio-Rad, Hercules, CA, USA). Acetone precipitated protein samples were reduced and alkylated with reagents supplied in the isobaric tags for relative and absolute quantitation (iTRAQ) kit (Applied Biosystems, Foster City, CA, USA). One hundred twenty µg of protein from each sample were digested with 1:40 enzyme to protein mass ratio of trypsin gold (Promega, Madison, WI, USA) overnight at 37 o C. The protein digests were desalted using Sep-Pak C18 cartridges (Waters, Milford, MA, USA) and dried under vacuum using a Speed Vac (Thermo Electron, Waltham, MA, USA). Peptides were labeled with the iTRAQ reagents according to the manufacturer's recommendations. MM peptides were labeled with tags 114 or 115 and MF peptides were tagged with 116 or 117 reporters respectively. A peptide mixture (for a total of about 400 µg of peptide digests) was made with all four tagged samples and desalted using Sep-Pak C18 cartridges to remove the detergents and excess iTRAQ reagents. Peptides were fractionated using a 3100 OFFGEL Fractionator by following manufacture guidelines (Agilent Technologies, Böblingen, Germany). 50 to 150 µl of fractions were recovered for each well and low peptide fractions from acidic (fractions 1-3) and basic (fractions 22-24) pI ranges were pooled. Peptides were then desalted in a Sep-Pak C18 cartridge, dried using a vacuum concentrator and resuspended in 10 µl of 0.1% formic acid. LC-MS/MS was performed as described above for 2-DE protein samples using a nanoflow UPLC (nanoAcquity, Waters) and ESI-hybrid Q-TOF (Premier, Waters, Milford, MA USA).

Protein identification and quantification
Protein identification and quantification followed procedures described in [10,17]. Micromass peak lists (pkl) containing the values from more than one spectrum are generated by a mass spectrometer's data handling system. A python script was applied to extract the reporter mass, i.e. 114-118 Da, from the non-deisotoping and deisotoping pkl files. The combined mass spectra pkl files were merged and searched against the concatenated "target" (real sequences of H. elegans (Hydroides) transcriptome) and "decoy" (reversed sequences of H. elegans transcriptome) translated sequences through search engine MASCOT version 2.3.0 (Matrix Sciences Ltd., London, UK). The search criteria were 30 ppm for precursor and 0.5 Da for fragments; ion scores no less than 20 at 95% confidence. Positive identification was matched to at least two unique spectra and had an E-value < 0.05. The false discovery rate (FDR) was set as less than 1%. Protein ratios were quantified based on the summed intensities of the spectrum matched. These ratios were log 2 transformed. Student's t-test and Benjamini and Hochberg correction were performed on the transformed ratio [18]. iTRAQ ratios that fulfilled the 1.3 fold criteria were considered significant. Proteins with expression ratios below 0.77 were considered to be down-regulated while those above 1.3 were considered up-regulated.

2-DE Western blot analysis
2-DE Western blot analysis for protein samples of MM and SF was performed to confirm the expression and distribution of the actin isoforms on 2-DE gels following the described protocol by Chandramouli et al. [9]. One hundred µg of protein were subjected to IEF on 7 cm IPG strips (Bio-Rad, Hercules, CA, USA), and separated on 12.5% SDS-PAGE. The proteins were transferred to PVDF membrane (Millipore Corporation, Bedford, MA, USA) and incubated (1:2000) with anti-actin clone C4 mouse monoclonal antibody (Millipore, Billerica, MA USA) for 14-16 hr at 4 o C. The membranes were then incubated with corresponding HRP secondary antibody followed by detection using a WesternBright ECL detection kit (Advansta, Menlo Park, CA, USA).

Identification of abundant protein spots
The reproducible abundant spots selected for protein identification are marked on 2-DE gels as shown in Figure 2 and Figure 3. In total, 36 proteins, of which 19 were phosphoproteins, were identified and listed in Table 1. The majority of the proteins appeared to be isoforms of the same protein. Several protein spots were identified either as myosin (spots 1-7, 12, 14) or non-muscle actin II (nm-ACT) (spots 15, 18, 19, 28, 25-27, 31-33 35) and actins (ACT) (spots 16,22,34,17,[20][21][22][23][24]. Spots 8 and 9 were identified as intermediate    (Figure 2). MF and SF proteome showed a lower degree of similarity in protein expression pattern. All other proteins were specifically expressed either in MF or SF with the exception of nm-ACTs which were present in both stages. IFs, GH, 14-3-3, TM, CaM, MLC, β-ACT, and ACT were specifically expressed in MF, whereas PDI, β-TUB, and FP were specific to SF (Figure 2). In contrast, males before and after spawning showed a higher degree of similarity in patterns. Both MM and MAS worms

Protein phosphorylation dynamics among MM, MAS, MF proteome
MHC, IFs, and MLC were frequently phosphorylated in MM, MAS, and MF. MHC, ACT, and β-ACT showed specific phosphorylation to MM, whereas phosphoprotein nm-ACT (spot 28) was specific to MAS. The phosphoproteome pattern between MF and SF was different. Twelve proteins or isoforms of the same protein, such as MHC, IFs, GH, 14-3-3, TM, CaM, MLC and ACT isoforms, were phosphorylated in MF, whereas only FP was phosphorylated in SF. In contrast, MM and MF were similar in phosphorylation patterns. Both male stages shared 12 (63%) phosphoproteins such as MLC, IFs, and ACT isoforms. Protein spots were treated with phosphatase to validate the specificity of phosphoproteins detected on 2-DE gels. The enzymatic treatment resulted in either downregulation or loss of phosphorylation of protein spots ( Figure 5). MHC (spots 1-6) and nm-ACTs (spots 18,19) were dephosphorylated and IFs (spots 8,9), ACT (spot 16), and β-ACT (spot 17) phosphorylation was down-regulated upon phosphatase treatment of ProQ Diamond stained gels ( Figure   5 right panel). These results validate the specificity of the phosphoprotein specificity of ProQ Diamond dye.

Differential proteins identified in MM and MF by gel-free quantitative proteomics
Threshold (E<0.05 and 1% FDR) and normalization parameters were applied for iTRAQ data analysis in order to eliminate the false positive protein identifications. Proteins were quantified using Student's t-test followed by Benjamini and Hochberg [18] correction and fold change cutoff for all iTRAQ ratios. In total, 19 differentially expressed proteins were identified, of which 10 were up-regulated and 9 were downregulated in MF when compared with those in MM. Table 2 lists all the proteins which showed statistically significant expression changes along with their fold-change values. Table S1 provides the raw MS data of all the proteins identified. Six myosin proteins including TP, MHC, troponin C (TC), and paramyosin (PM) were down-regulated in MF worms. Two translation related proteins, such as ribosomal protein S2 (RP2) and elongation factor 1-alpha (EF1), were up-regulated. Six uncharacterized or partially characterized proteins were upregulated. Four proteins, such as guanine nucleotide-binding protein (G-protein) and TUB, were also up-regulated, whereas ATP synthase and ACT were down-regulated (Table 2).

Validation of abundant actin isoforms on 2-DE gels of MM and SM
Commercial antibodies against polychaetes proteins are not available. Fifty two percent of the identified proteins were isoforms of ACT and showed different molecular mass (Mr) and isoelectric point (pI) values. Conserved protein actin was used to validate its abundant occurrence of isoforms on-2-DE gels. ACT (spots 16,21,22) and β-ACT isoforms (spots 17, 20, 24) had MW 25-10 and pI values 5.0-6.0 ( Figure 6; left panel) in MM. SF showed specific isoforms of nm-ACTs (spots [31][32][33]35) with Mr and pI valves ranged from 66-40 and pI values 5.5-6.5, respectively ( Figure 6; right panel). Although ACT is highly conserved and expressed in male and female worms, the occurrence of different sets of its isoforms in MM and SF indicates the different functional and structural roles during reproduction in Neanthes.

Proteomic changes in males before and after spawning
No previous studies have been done to distinguish molecular events during unusual reproductive behavior in male and female worms of polychaetes. This proteomic study provides molecular insights in protein expression pattern and their phosphorylation status during the reproductive period. Growth is usually measured by the number of segments in Neanthes [2]. The new segments may be added even 10 days before spawning. The occurrence of higher number of proteins in mature males before spawning may be attributed to a high demand of protein synthesis that is probably required for body growth and segment formation. In MM, the specific expression of ACT and nm-ACTs isoforms before and after spawning indicates their different functional and structural roles during reproductive development. For example, after fertilization the male undulates his body continuously to pump clean water and to expel metabolic wastes while incubating developing embryos [3], the nm-ACTs isoforms possibly facilitate the morphological movement of his body. The down-regulation of myosin and 14-3-3 proteins after spawning may indicate the sign of ageing which results in weakening of the body. MM fights off both sexes by utilizing most of his energy to safeguard the eggs during fertilization and development [2]. In MM, the specific expression of GH might facilitate the energy production from glycolytic pathway and its subsequent feedback control by CaM signaling molecules. GH is the major catalytic enzyme for the synthesis and breakage of glycosidic bonds [19]. The CaM is a multifunctional messenger protein that transduces calcium signals and interacts with various target proteins to mediate many biological processes [20].

Protein Phosphorylation Dynamics in Males before and after Spawning
The proteomic changes that render mature males able to fertilize the eggs have been correlated with the changes in phosphorylation of several proteins. In a previous study, we identified tyrosine/serine phosphorylation of proteins in early larvae of Neanthes [9]. Identification of 19 phosphoproteins that exhibited gender/ stage specific phosphorylation suggests that the proteins, which influence reproductive changes, may be regulated by its phosphorylation changes (e.g. phosphorylation of specific set of proteins). For instance, phosphorylation of MLC and ACT isoforms were specific to MM, whereas 14-3-3 and TM showed specific phosphorylation after spawning. Despite these differences in specific phosphorylation, both MM and MAS worms shared 12 (63%) phosphoproteins (MLC, IFs, and ACT isoforms) that did not change expression. These common phosphoproteins, expressed before and after spawning, were probably required for the housekeeping functions during reproduction of the male [12]. The phosphorylation of MLC displays multi-functions in muscle tissues as well as non-muscle cells [21,22]. For example, cAMP-dependent kinase phosphorylates regulatory light chain isoform of MLC and modulates the kinase activity [23].
Transcripts of MLC and TM were shown to be involved in muscle development in competent larvae of H. elegans [24]. In previous study, we showed that the abundant expression of Ca 2+ /CaM dependent MLC kinase is crucial to larval settlement of the barnacle Balanus amphitrite [25]. In polychaete H. elegans, CaM gene was abundantly expressed in growth zones, branchial rudiments, and collar regions of competent larvae and juveniles [26]. The phosphorylation of IM and ACT isoforms may play a critical role in regulating the function of actin filaments of muscle and non-muscle cells [27]. Based on these findings, we deduced that phosphorylation of myosin and cytoskeleton proteins are an essential process that may influence developmental and reproductive changes in Neanthes.

Changes in protein expression and phosphorylation in spent female
The body muscles of the female are reabsorbed to supply the nutrients for the large yolky eggs, which results in a loss of 75% of her body weight [2]. The decrease in protein (21%) and phosphoprotein spots (70%) followed by low levels of phosphorylation may be attributed to protein degradation that had lead to the loss of body weight after egg laying [3]. The stress response (PDI) and hence metabolism (FP) proteins may be required to evade the immune system in response to weakening of body. The complete down-regulation of ACT isomers and IFs in SF may be attributed to the degenerating architecture of the cytoskeleton, which subsequently leads to breakdown of cellular processes. Myosins composed of actindependent molecular motor proteins are responsible for actinbased motility in skeletal muscle [28]. All eukaryotic cells composed of myosin isoforms have specialized functions in specific cell types such as muscle [29]. MHC, MLC, TM, TC, and PM proteins are highly conserved motor proteins and contribute to muscle contraction and motility processes [30].  Down-regulation of myosin in spent females may have affected the muscular contraction and thereby reduced their locomotive ability [2]. Furthermore, myosin function in cellular pathways is mediated by its interaction with specific proteins and is controlled by regulatory signals and protein phosphorylation [23,28]. For example, the contraction of skeletal and cardiac muscles is regulated by the coordinated interaction of myosin filaments with regulatory molecules such as Ca 2+, CaM, TC, and ACT that often act as an ON/OFF switch that activates or deactivates muscular contraction [31][32][33]. The most important finding of this study is the dephosphorylation reductions of ACT and MHC isoforms in SF. MF exhibited a high degree of phosphorylation of ACT and MHC isoforms whereas only FP was phosphorylated in SF. Furthermore, MF may have synthesized most of the abundant proteins that are subsequently phosphorylated. These proteins may serve as an energy source for maturing eggs. These results indicate that phosphorylation or dephosphorylation of specific proteins influences reproductive changes in females.

Gender specific differential expression of proteome in MM and MF worms
The morphological and behavioral difference such as egg bearing female and egg incubating males may be influenced by differences in their proteome expression patterns that play an important role in reproduction [12]. Proteomes of MM and MF were less divergent before and after fertilization with the exception of changes in MHC (spots 1-7) and phosphorylation of ACT isoforms (spots 15, 20, 21) in 2-DE profiles. Similarly, MM and MF revealed similar trend of expression pattern of phosphoproteome. We believe that prior to spawning immature MM and MF exhibit common morphological features that are associated with the same set of proteome. In previous study [9], we proposed that 2-DE proteome analysis of whole worms increases sample complexity and thus prevents identification of low abundant proteins [34,35]. In addition, 2-DE proteomics generates proteome profiles that only represent highly abundant proteins [36,37]. These limitations accounted for less divergent 2-DE proteome profiles of MM and MF worms. To obtain comprehensive proteomics changes between MM and MF worms, we used quantitative proteomics that led to identification of 19 differentially expressed proteins. MM and MF worms showed differences in the levels of expression of proteins that are known to be involved in the cell signaling, structure and integrity, translation, and muscle contraction. These proteins possibly involved in signaling or sensing activities upon pairing and their subsequence reproductive changes between male and females. Down-regulation of MHC in MF is in agreement with the 2-DE proteomics data. In SF worms the lining of the digestive tract remains intact and body muscles undergo autolysis resulting in accumulation of phagocytized fragments of muscle tissue [38,39]. Eleocytes are produced in large numbers in both sexes shortly before and after the spawning [40]. Muscle fragments are used for the synthesis of vitellogenin, yolk precursor in oocytes [41]. In N. arenaceodentata, the lipid and protein needed for vitellogenin synthesis appear to be provided from muscle tissue degradation by coelomocytes [6]. Identification of several myosin fragments or isoforms in MM, MF, and MAS would further support this argument. The myosin function appears to be regulated in a number of ways; for example, by the presence or absence of phosphorylation, by Ca 2+ /CaM binding, and/or by binding to ACT, TC, and TM, which may synergistically modulate the reproductive events [31][32][33]. The up-regulation of G-proteins and translation proteins may indicate the synthesis and storage of new proteins that may be capable of converting chemical energy into mechanical work nutrients for the maturing eggs. Chaperone proteins (HSP90s) are flexible dimer ATPases that bind to receptors, transcription factors, and protein kinases are important for development and survival [42]. The reduced level of ATPase expression, which is capable of converting chemical energy into mechanical force, may also indicate the weakening of the female body. GL12416, hypothetical protein, and unnamed predicted proteins which were up-regulated in MF have no known function. 2-DE proteomics identified several proteins such as MHC, ACT, and TUB that constitutively expressed both in male and female worms. This pattern of observation is also consistent with the gel-free quantitative proteomics data suggesting that both techniques are complementary. The abundant expression of similar proteins in P. vexillosa and H. elegans may suggest that polychaetes maintain a pool of abundant proteins of housekeeping or regulatory functions [43,44].

Validation of phosphoprotein spots and distribution of actin isoforms on 2-DE gel
Phosphatase treatment of protein spots 2-DE gel confirmed the phosphorylation of several proteins. This finding allowed us to speculate that MHC and ACT isoforms are probably involved in post-translational regulation, such as phosphorylation and dephosphorylation. The results of this experiment also confirmed the phosphorylation of several proteins on 2-DE gels. The results of 2-DE Western-blot confirmed the specific expression patterns of different isoforms in male (ACTs) and female (nm-ACTs) worms. These results suggest that although ACT is highly conserved in male and females, isoforms of certain ACT may play the same or different functional roles in modulating reproductive behavior in polychaetes. Overall, the validations experiments supports the hypothesis that protein modification (phosphorylation) dynamics influences reproductive pattern by activating (phosphorylation) or deactivating (dephosphorylation) protein's function.

Conclusion
We demonstrated gel-based and gel-free proteomic methods are feasible to document the proteomic differences between male and female worms before and after spawning of the polychaete worm Neanthes. The varieties of isoforms of myosin's and actins may interact each other and influence the behavioral and morphological differences between sexes. The identified proteins involved in muscular development, cell signaling, structure and integrity, and translation were differentially expressed and probably regulate reproductive behavior. The proteome dataset of the male and female worms shall be an important resource for further understanding the unusual reproductive pattern in Neanthes acuminata complex in which the female reproduces once and the male is capable of reproducing many times [3]. Table S1. Raw MS data of all the proteins identified by gelfree quantitative proteomics. (XLSX)