Proteomics reveals the preliminary physiological states of the spotted seal (Phoca largha) pups

Spotted seal (Phoca largha) is a critically endangered pinniped in China and South Korea. The conventional method to protect and maintain the P. largha population is to keep them captive in artificially controlled environments. However, little is known about the physiological differences between wild and captive P. largha. To generate a preliminary protein expression profile for P. largha, whole blood from wild and captive pups were subjected to a label-free comparative proteomic analysis. According to the results, 972 proteins were identified and predicted to perform functions related to various metabolic, immune, and cellular processes. Among the identified proteins, the expression level of 51 were significantly different between wild and captive P. large pups. These differentially expressed proteins were enriched in a wide range of cellular functions, including cytoskeleton, phagocytosis, proteolysis, the regulation of gene expression, and carbohydrate metabolism. The abundances of proteins involved in phagocytosis and ubiquitin-mediated proteolysis were significantly higher in the whole blood of wild P. largha pups than in captive individuals. In addition, heat shock protein 90-beta, were determined as the key protein associated with the differences in the wild and captive P. largha pups due to the most interactions of it with various differentially expressed proteins. Moreover, wild P. largha pups could be more nutritionally stressed and have more powerful immune capacities than captive pups. This study provides the first data on the protein composition of P. largha and provides useful information on the physiological characteristics for research in this species.


Results
A total of six whole blood samples from P. largha pups (three from wild pups and three from captive pups) were measured using label-free proteomic analysis. After data filtering and protein identification, a total of 4562 unique peptides were obtained and 972 proteins were identified from P. largha pup blood (Supplementary file 1). To investigate the physiological functions of P. largha pups, annotations from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were extracted based on the reference transcripts that matched the identified P. largha proteins. According to the results of GO annotations, the major protein functions included cellular process, metabolic process, biological regulation, and response to stimulus belonging to the biological process aspect, intracellular and cellular anatomical entity in the cellular component aspect, and binding and catalytic activity attribute to the molecular function aspect (Fig. 1a). For KEGG pathway analyses, the identified proteins exhibited a broad functional distribution, among which the dominant pathways were metabolic pathways, and the complement and coagulation cascades (Fig. 1b).
Based on the identified proteins, partial least squares discrimination analysis (PLS-DA) was used to comprehensively investigate the divergence of the preliminary protein expression profiles between wild and captive P. largha pups (Fig. 2). The first two components explained 57.8% of the total protein expression variations, and samples from the wild and captive individuals were observed in isolated clusters, separated from one another. Those results suggested that captivity in artificial environments could significantly affect the protein composition and abundance in the whole blood of P. largha pups. Furthermore, the expression of 51 proteins exhibited significant variations between the whole blood of wild and captive P. largha pups (Supplementary File 2). Compared to the samples from captive pups, the number of up-regulated DEPs in the wild-pup samples was 26 (Table 1),   Table 2). Moreover, there were eight and ten proteins unique to the wild-and captive-pup samples, respectively (Table 3). Thus, those results revealed the dissimilarity in the protein expression profiles in whole blood from wild and captive P. largha pups. As illustrated in the DEP tables, some important immune-related proteins, such as Cathepsin S (CTSS), Protein phosphatase 1A (PP1A), CD59 glycolicprotein (CD59), Protein phosphatase type 2A (PP2A), Heat shock protein HSP 90-beta (HSP90AB1), and Calpain-1 catalytic subunit (CAPN1) were up-regulated in the blood of wild P. largha. In contrast, five proteins related to immune response were up-regulated or uniquely expressed in captive pups, including Glutathione S-transferase theta-1 (GSTT1), Vitamin K-dependent protein S (PROS1), Protein DDI1 homolog 2 (DDI2), Immunoglobulin alpha heavy chain (IGHA), and Galectin-3-binding protein (LGALS3BP). Moreover, several proteins involved in the regulation of gene expression were significantly upregulated in the blood of wild P. largha pups, including Barrier to autointegration factor 1 (BANF1), NIF3-like protein 1 (NIF3L1), Calcium-regulated heat stable protein 1 (CARHSP1), Eukaryotic translation initiation factor 5 (EIF5), and Exportin-1 (XPO1). It is also worth noting that the expressions of multiple proteins related to cell adhesion were differentially expressed in the blood of wild P. largha pups, including Filamin A (FLNA), Tubulin beta chain (TUBB), Vinculin (VCL), Ankyrin-3 (ANK3), Translationally-controlled tumor protein 1 (TPT1), Protein argonaute-2 (AGO2), F-actin-capping protein (CAPZA1), Vasodilator-stimulated phosphoprotein (VASP), and Fibrinogen alpha chain (FGA).
We also performed GO and KEGG enrichment analyses to explore the biological functions and pathways that could be significantly impacted due to captivity. The results showed that 13 GO terms were significantly enriched in the DEPs between wild and captive P. largha pups (Fig. 3). The enriched GO terms were assigned to three classes: (1) cell structure, including different kinds of organelle and cytoskeleton functions; (2) regulation of translation, which involves a broad range of post transcriptional and translational regulations; and (3) others, containing monosaccharide and carbohydrate binding, and cellular and chemical homeostasis.
KEGG pathway enrichment analyses based on the DEPs revealed that ten pathways were significantly enriched in the blood of wild and captive P. largha pups (Fig. 4). According to the function of the enriched pathways, they were classified into three categories, including phagocytosis, proteolysis, and carbohydrate metabolism. The phagocytosis category included Fc gamma R-mediated phagocytosis and its related cell adhesion pathways. The proteolysis category included ubiquitin-mediated proteolysis and its accompanying signaling pathways. The carbohydrate metabolism category included the pentose, galactose, fructose and mannose metabolism pathways.
The protein-protein interaction (PPI) networks of the identified DEPs were analyzed using STRING software and the results are shown in Fig. 5. Based on the search results, HSP90AB1 had the most predicted interactions with the other DEPs. A variety of cell adhesion and cytoskeleton proteins (VASP, VCL, and FGA, among others) was predicted to interact with HSP90AB1 via FLNA. Additionally, HSP90AB1 was predicted to associate with a series of post-transcriptional (XPO1, AGO2 and NIF3L1) and translational regulation (RPLP1, EIF5, TPT1, etc.) proteins through its interactions with AHCY and TUBB, respectively. Moreover, the interaction between Table 2. Down-regulated proteins in the whole blood of wild Phoca largha pups compared to captive pups.  Table 3. Unique proteins in the whole blood of wild and captive Phoca largha pups.  www.nature.com/scientificreports/ HSP90AB1 and PSMA2 was associated with proteins involved in ubiquitin-mediated proteolysis (UBE2O, UBA1 and UBA7).

Discussion
Until now, no studies have been performed on the variations in protein composition and expression in P. largha.
One of the aims of the current study was to determine the preliminary proteomic profile of P. largha. A total of 972 proteins were identified using label-free proteomics analyses. Regarding other marine mammals, a previous study of kidney diseases in wild animals identified a total of 2694 proteins in the urine of 19 California sea lions (Zalophus californianus) 21 . However, only 206 proteins were identified from the cerebrospinal fluid of 11 California sea lions 22 . Conversely, two previous studies of plasma samples from bottlenose dolphins (Tursiops truncates) only identified 58 and 196 unique proteins, respectively 23,24 . Thus, those results suggested that the complexity of the protein compositions of marine mammals varies between different tissues and species. Another aim of this study was to identify the proteins that were differentially expressed between the whole blood of wild and captive P. largha. In the present study, the Fc gamma R-mediated phagocytosis pathway was enriched in the blood of wild and captive P. largha pups. Phagocytosis is an important innate immune defense mechanism in animals, and is accompanied by a chain of cellular processes as diverse as cytoskeletal rearrangement, alterations in membrane trafficking, and the activation of microbial killing mechanisms 25 .
Several cytoskeleton-related proteins were found to be up-regulated in the blood of wild P. largha pups compared to captive pups, including VASP, FLNA, TUBB, VCL, and CAPZA1. VASP is an actin-associated protein that promotes actin filament elongation 26 . The activation of Fc gamma receptors during phagocytosis triggers the recruitment of VASP to phagosomes 27 . FLNA is an actin-binding protein that anchors various transmembrane proteins to the actin cytoskeleton to promote cell-cell contact 28 . FLNA has been reported to play a role in neutrophil phagocytosis in humans 29 , and the deletion of FLNA resulted in a twofold reduction in collagen phagocytosis in mice 30 . The rate-limiting step during phagocytosis is the binding of specific adhesion receptors, such as integrins 31 . TUBB and VCL are intracellular adaptor proteins that link actin filaments and integrins to construct the cytoskeleton 32,33 . CAPZA1 performed to the fast-growing ends of actin filaments and its mutation Figure 5. Protein-protein interaction (PPI) networks for differentially-expressed proteins between the whole blood of wild and captive Phoca largha pups. The PPI network had an average node degree of 1.35 and clustering coefficient of 0.491. The PPI enrichment p-value was 0.00532, which indicated that the network had significantly more interactions than expected. The colors of edges specify different types of interaction evidences and the thickness indicates the confidence of data support. STRING 20 (https ://strin g-db.org/) was used to construct the PPI networks of the DEPs to identify the key proteins that changed the physiological functions of P. largha due to captivity.

Scientific Reports
| (2020) 10:18727 | https://doi.org/10.1038/s41598-020-75759-2 www.nature.com/scientificreports/ resulted in actin accumulation, thereby blocking cytoskeletal rearrangement 34 . Conversely, FGA, an extracellular protein that interacts with integrins for cell-cell adhesion 35 , was uniquely detected in the blood of captive P. largha. It has been shown that the binding of FGA by Streptococcus progenies was involved in their resistance to phagocytosis in human blood 36 . Thus, all of the above findings suggest that the blood of wild P. largha pups contained more phagosomes than that of captive pups. Moreover, during phagocytosis, macrophages alter the plasma membrane to form the phagosome, which is the principal site for killing microorganisms through proteolysis 37 . In the present study, proteins involved in the ubiquitin-mediated proteolysis pathway were enriched in the blood of P. largha pups, including UBE2, UBE2O, UBA1 and ATG3. The activation of ubiquitin-mediated proteolysis requires ATP to degrade microbial membrane proteins 38 . G6PDX, which catalyzes the rate-limiting step of the oxidative phosphate pathway to produce ATP 39 , was up-regulated in the blood of wild P. largha pups.
In summary, the DEPs involved in phagocytosis and proteolytic activities detected in the present study suggest that wild P. largha pups may be more resistant to pathogen infection than captive pups. The heightened level of immunity may be due to the result of ontogenetic differences between the two groups. In a species with such rapid post-natal development, animals that are < 1 month of age (wild pups in this study) are going to have dramatically different proteome profiles than animals that are 4 months of age (captive pups in this study). Similar to humans, newborn pups will acquire more powerful protection via maternal passive immunity from their mothers to ensure their survival 40 , and the immunity inherited from the mother could not have been completely degraded in the detected wild pups. Moreover, captive pups lived in relatively stable environments and were cared for by humans; thus, they were likely to be less environmentally stressed than wild pups. Such factors could be additional reasons for the higher expression of proteins associated with immune function of wild P. largha pups compared to their captive counterparts.
The biological roles of the DEPs identified in the present study were further examined based on their PPI networks to understand differences between wild and captive P. largha pups. HSP90AB1, a type of heat shock protein was upregulated in the wild P. largha pups, and was predicted to interact with a variety of DEPs of different functions, including those involved in cell adhesion, regulation of gene expression, and proteolysis. This result illustrated that HSP90AB1 could be the key protein related to the differences in the protein expression profiles between wild and captive P. largha pups. The realization of HSP90AB1 physiological functions depends on the consumption of ATP. Upon ATP binding, HSP90 undergoes significant conformational changes to become active and the ATP was hydrolyzed to ADP after the activation of client proteins 41 . This phenomenon was consistent with the up-regulation of proteins involved in carbohydrate metabolism in the blood of wild P. largha pups; thus, indicating that there might be more energy demand. Spotted seal pups in the wild are born in pack ice, which exposes them to higher predation than other animals. The pack ice breeding strategy resulted in the evolution of shorter lactation times and higher daily energy outputs in spotted seals 42 . In captive environments, newborn spotted seal pups are weaned and regularly fed. However, even if they have begun foraging, wild pups are likely to be more nutritionally-stressed than captive pups. On the other hand, wild pups are likely to respond differently to presence of and handling by researchers than pups in captivity. Sampling may induce a more robust physiological stress response in wild pups than captive pups, resulting the alteration of protein expression. Thus, the potentially higher nutritional and physiological stresses could be the underlying reason for the upregulation of proteins involved in carbohydrate metabolism and immunity in the blood of wild P. largha pups.
Due to ecological destruction and poaching, the number of spotted seals in Liaodng Bay, China is very low. Field rescue and captive breeding are the dominant programs for maintaining the Liaodong Bay population of spotted seals. The simultaneous occurrence of on-ice births, weaning pups, and ice-melting are important characteristics for the reproductive biology of these seals 43 . Increased ice melting due to global warming and harsh sea conditions have caused high death rates among spotted seal pups 42 . Therefore, higher expression of proteins associated with carbohydrate metabolism in wild compared to captive pups may be the result of higher metabolic demands of postnatal development in wild seals. Conversely, spotted seal pups born in aquariums are susceptible to diarrhea and pneumonia, both of which are caused by pathogenic bacteria 44,45 . The diseases of spotted seal pups in captive environments can lead to anorexia and death in severe cases. The downregulation of proteins associated with phagocytosis and proteolysis detected in the present study was consistent with high incidence of disease in captive pups. Therefore, it is necessary to improve the immunity of captive spotted seal pups through food additives or other methods. In addition, while proteomes of spotted seal adults were not measured currently, the results of spotted seal pups in this study are likely to differ from the adults. The data described here only provide preliminary insights into the physiology of this species, specifically in pups, but are by no means a comprehensive explanation of the impact of captivity on ringed seal physiology. In conclusion, the information provided herein not only expands the understanding of protein expression profiles in spotted seals, but also provides information for the conservation of this species.

Methods
The wild P. largha pups investigated in this study were sampled from Liaodong Bay, China, during the Spotted seal Rescue Survey in 2019. Based on the white lanugo on their skin, and the fact that they were independent of their mother indicated that they ended lactation, but were less than one month of age. The captive P. largha pups used in this study were from the Dalian Sun Asia Aquarium (DSAA), China, and were all newborn under human care in 2019. The captive pups were weaned and were regularly fed prior to sampling. The diet of the captive spotted seals was a 1:1 ratio of capelin Mallotus villosus and Atlantic herring, Clupea harengus (m:m). The amount of feed was approximately 8% of the pup's weight per day. The ontogenetic differences of wild and captive P. largha pups are provided in Table 4. Approximately 3 mL of blood was collected from the veins in the hind flippers when the animals were restrained on a V-shaped bench. The handling protocol was the same for wild and Scientific Reports | (2020) 10:18727 | https://doi.org/10.1038/s41598-020-75759-2 www.nature.com/scientificreports/ captive pups. Blood samples were stored in medical biochemical tubes with anti-coagulation gel at − 80 °C prior to protein extraction. All operations were performed in accordance with the relevant guidelines and regulations. Label-free proteomics technology was used to generate the preliminary whole blood protein expression profiles of wild and captive P. largha pups. Proteins from the whole blood samples were extracted using the Mammalian Tissue and Cell Protein Extraction Kit (Bangfei Bioscience Co., Ltd, Beijing, China). The concentrations of protein in each sample were determined using a protein quantification kit (Dingguo Changsheng, Beijing, China), according to the manufacturer's instructions. The extracted proteins were then excised from the preparative tube and destained with NH 4 HCO 3 . Following reduction and alkylation with dl-dithiothreitol and iodoacetamide, respectively, the samples were digested with trypsin using the Filter Aided Sample Preparation protocol 46 . All digested peptide samples were stored at − 80 °C prior to MS analysis.
MS analysis was performed according to a previous study with only one technical replicate for each sample 47 . Digested peptide mixtures were first pressure-loaded onto a fused silica capillary column packed with 3 μm dionex C18 material (Phenomenex, USA). The column was washed with buffer A (water, 0.1% formic acid) and buffer B (acetonitrile, 0.1% formic acid), and an Agilent 1100 quaternary high-performance liquid chromatography (HPLC) was applied to analyzed. The first step of HPLC measure was consisted of a 5 min gradient from 0 to 2% buffer B, followed by a 45 min gradient to 40% buffer B. Following this, a 3 min gradient from 40 to 80% buffer B was performed followed by a 10 min hold at 80% buffer B. A 2 min gradient of buffer B from 80 to 2% was performed, and approximately 100 μg of the tryptic peptide mixture was loaded onto the columns and was separated at a flow rate of 0.5 μL/min using a linear gradient. As peptides were eluted from the micro-capillary column, they were electrosprayed directly into a micrOTOF-Q II mass spectrometer (BRUKER Scientific, Beijing, China) with the application of a distal 180 °C source temperature. The mass spectrometer was operated in the MS/ MS mode. Survey MS scans were acquired in the TOF-Q II with the resolution set to a value of 20,000. Each survey scan (50-2500) was followed by five data-dependent tandem mass scans at a normalized scan speed of 2 Hz.
Tandem mass spectra were searched against the reference transcriptome of spotted seals (NCBI Sequence Read Archive: SRA050171) 1 using the Proteome Discoverer 2.1 software by the Mascot search engine (Matrix Science, London, UK; version 2.3.02) based on the standard LFQ module. The following options were used to identify the proteins: Peptide mass tolerance = ± 15 ppm, MS/MS tolerance = 0.02 Da, enzyme = trypsin, missed cleavage = 2, fixed modification: carbamidomethyl, variable modification: oxidation, database pattern = decoy. The results were then filtered using a cutoff of 0.01 for the false discovery rate. The minimum number of peptides required to identify a protein was set to 1 and proteins with at least two unique peptides were used for abundance quantification. The quantification of peptides was based on MS1-level data. For differential expression analysis, proteins with missing abundance values in more than one biological replicate of wild or captive groups were filtered out. If a protein with more than one abundance values in one groups and missing abundance values in all three replicates in another group, it was identified as unique in this group. Then, remaining proteins with fold changes > 1.5 and p-values < 0.05 (t-test) between the wild and captive P. largha pups were considered to be significantly differentially expressed.
To determine the biological functions of the preliminary whole blood proteome of P. largha pups, the GO and KEGG annotations of the identified proteins were extracted from the annotation results of the reference transcriptome. Such annotations were obtained from our previous study by aligning the unigenes to the GO and KEGG databases 48 using BLASTx with an E-value cutoff of less than 10 -51 .
PLS-DA based on the identified proteins and their expression levels in all samples was performed using the "mixOmics" package v6.12.1 49 in the R v4.0.0 platform to evaluate the differences in whole blood protein expression profiles between the wild and captive P. largha pups. The GO and KEGG enrichment analyses were performed using the DAVID tool based on the GO and KEGG annotations of DEPs 50 , and the p-values were adjusted using the FDR method. STRING 20 was used to construct the PPI networks of the DEPs to identify the key proteins that changed the physiological functions of P. largha due to captivity. All other charts were drawn using the "ggplot2" package in R v4.0.0 software.