The mud crab Scylla paramamosain, one of a number of commercially important crustaceans, is widely distributed along the coast line of southern China and the broader Pacific area. Little is known about the disease burden of these animals in the juvenile or adult phases of S. paramamosain, but susceptibility to infection has been identified during the larval stages. Several bacterial and viral pathogens have been reported to infect crabs (1). At present, bacterial infections are the main cause of crab disease and constraint in the crab farming industry in China (2, 3). In aquaculture, Vibrio alginolyticus is an important bacterial pathogen that would cause huge economic losses and has caused many cases of natural infection in the Chinese mitten crab (4). White spot syndrome has become the most hazardous and devastating disease in shrimp culture (5). White spot syndrome virus (WSSV) can also infect several species of crab both in the natural and experimental setting (6–8).

As we know, crustaceans including crabs only have an innate immune system to defend against invading microbes (9). The innate immune system includes phagocytosis, encapsulation, melanization, coagulation, and the release of antimicrobial peptides (AMPs) in crustaceans (9, 10). The hemocytes are the main site in crustaceans where the immune defense is mounted. Proteomics have been widely used to investigate the molecular mechanisms underlying the immune response to WSSV infection in crustaceans but there is no research available on V. alginolyticus infection yet (11, 12). Currently an isobaric tag-based methodology for peptide relative quantification [isobaric tags for relative and absolute quantitation (iTRAQ)] coupled to multidimensional liquid chromatography and tandem mass spectrometry enables the assessment of protein levels where four samples can be compared for their common effects (13). iTRAQ analysis has been performed to reveal the effects of Spiroplasma eriocheiris infection in Chinese mitten crab (Eriocheir sinensis) hemocytes (14). Crab hemocytes have been confirmed as the major target cells in WSSV or V. alginolyticus infection. The aim of this study was to investigate the hemocytes’ immune response to WSSV or V. alginolyticus infection at the protein level.

The iTRAQ analysis of S. paramamosain hemocytes proteome showed 22, 834 queries in the database (314,202 sequences) and 1,799 proteins were identified from LC–MS/MS spectra and peptides (Figure 1A). The protein mass distribution showed that protein mass above 100 kDa occupied 16% and protein mass between 10 and 60 kDa occupied 59% (Figure 1B) of the identified proteome. In an effort to analyze the functional distribution of the identified proteins, the known proteins from S. paramamosain hemocytes were used as a reference in the COG database (Figure 1C). The functional classification of the S. paramamosain sequences showed that the most common function was R: general function prediction only, which indicated that many sequences had an unknown function. The second function was O: posttranslational modification, protein turnover, chaperones, the third function was J: translation, ribosomal structure, and biogenesis. KEGG pathway classification showed that the major organismal system was the metabolism that contained 226 identified proteins, and the major metabolic pathway was carbohydrate metabolism (109 proteins). The second pathway was translation (212 proteins), which represents genetic information processing, and the third pathway was transport and catabolism (160 proteins), which represents cellular processes. The cellular component identified by GO analysis is depicted in Figure 2A. GO analyses showed that the peptides can be categorized into several biological process, i.e., cellular process (18.67%), metabolic process (17.50%), single-organism process (10.37%), and the following two regulatory stages: biological regulation (6.82%) and regulation of biology processes (6.41%) (Figure 2B). The major molecular functions identified by GO analysis of the peptides were binding (44.66%) and catalytic activity (42.14%) (Figure 2C). The KEGG pathway classification of proteins in hemocytes from healthy crabs showed that metabolic pathways and translation are very important (Figure 2D).

The crabs were challenged with WSSV or V. alginolyticus, and then the samples were used for iTRAQ analysis and compared with the control group. We applied a 1.2-fold (P < 0.05) increase or decrease as a threshold for significant differences in protein expression to qualify a physiologically important change, 321 proteins were shown to meet this criteria for differential expression, including 157 upregulated proteins and 164 downregulated proteins after WSSV infection (Figures 3A,B). A further 243 proteins showed a 1.2-fold (P < 0.05) increase or decrease in protein expression during V. alginolyticus infection including, 121 upregulated proteins and 122 downregulated proteins. WSSV infection affected more protein expression in crab hemocytes than V. alginolyticus infection. Interestingly, 106 differentially expressed proteins were common to both infections (Figure 3B). Of these, copine-8, troponin C isoform 2b, and sarcoplasmic calcium-binding protein 1 were upregulated significantly, and Sacsin, ALF 4, ALF iso4, and Pseudomonas aeruginosa pathogenicity island (PAPI) I were downregulated significantly (Tables 1–3). The data indicate that these proteins may participate in the immune response to WSSV infection and V. alginolyticus infection. These 106 proteins can be categorized into some important pathways including carbon metabolism, ribosome, mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, phagosome, hippo signaling pathway among others (data not shown). The major molecular functions determined by GO analysis for these proteins were binding, catalytic activity, and organelle components (Figure 3C).

Of the differentially expressed proteins in WSSV infection, the GO enrichment scatterplot showed that the organelle and intracellular organelle proteins were the most important, and a KEGG enrichment scatterplot indicated that metabolic pathways were the most important (Figure 4; Figure S1A in Supplementary Material). The differentially expressed proteins (over twofold) and the related iTRAQ data are presented in Table 1. The upregulated proteins included copine-8 with 6.29-fold change, troponin C isoform 2b with 5.35-fold change, sarcoplasmic calcium-binding protein 1 with 4.78-fold change, beta-actin with 4.02-fold change, myosin light chain 2 with 3.58-fold change, slow muscle myosin S1 heavy chain with 2.69-fold change, clathrin light chain with 2.67-fold change, ubiquitin carboxyl-terminal esterase L3 with 2.65-fold change, 2-phospho-d-glycerate hydrolase with 2.65-fold change, membrane-associated protein with 2.64-fold change, alpha actin with 2.56-fold change, myosin-9 with 2.1-fold change, myosin heavy chain type 1with 2.04-fold change and some enzymes with above a twofold change (Table 1). The data indicate that many endocytosis-related cytoskeleton proteins of host hemocytes contribute to WSSV infection. The upregulated proteins and the related iTRAQ data are presented in Supplementary Material Table S1. The downregulated proteins included Sacsin with 8.76-fold change, pacifastin-like serine protease inhibitor with 5.45-fold change, male reproductive tract-specific Kazal-type proteinase inhibitor with 3.77-fold change, PAPI-1 with 3.1-fold change, ALF 4 with 2.59-fold change, crustin AMP with 2.47-fold change, heat shock protein 90 with twofold change and some enzymes with above a twofold change.

Of the differentially expressed proteins in V. alginolyticus infection, the GO enrichment scatterplot showed that the organelle and intracellular organelle are the most important, and a KEGG enrichment scatterplot indicated that metabolic pathways were the most important (Figure 5; Figure S1B in Supplementary Material). The differentially expressed proteins (over twofold) and the related iTRAQ data are presented in Table 2. The upregulated proteins included sarcoplasmic calcium-binding protein 1 with 8.4-fold change, troponin C isoform 2b with 2.78-fold change, clip domain serine proteinase 3 with 2.4-fold change, putative secreted salivary gland peptide with 2.23-fold change, copine-8 with 2.17-fold change, slow muscle myosin S1 heavy chain with 2.13-fold change, chloride intracellular channel isoform 1 with 2.11-fold change, and myophilin with 2.03-fold change (Table 2). The downregulated proteins included Sacsin with 31.39-fold change, phospholipase D1 with 6.97-fold change, low-density lipoprotein receptor with 5.77-fold change, PAPI I with 3.02-fold change, pacifastin-like serine protease inhibitor with 2.99-fold change, ALF 4 with 2.82-fold change and ALF iso4 with 2.12-fold change. It was showed that intracellular non-membrane-bounded organelle is the most important in statistics of GO enrichment of differentially expressed proteins (more than 1.2-fold) from WSSV vs V. alginolyticus, and phagosome is the most important in statistics of GO enrichment (Figure S2 in Supplementary Material).

Interestingly, some proteins were changed significantly (P < 0.01) in WSSV and V. alginolyticus infection (Table S2 in Supplementary Material; Table 3). Six differentially expressed genes, β-actin, myosin-9, ALF iso4, ALF 4, transketolase-like protein 2-like isoform 1, and sarcoplasmic calcium-binding protein 1 were characterized by real-time PCR assay (Figures 6A,B). The expression of these six genes showed a trend of upregulation at 24 h post WSSV or V. alginolyticus challenge except for that of myosin-9 in response to WSSV. Only β-actin and sarcoplasmic calcium-binding protein 1 showed the same trend in the proteomic results. dsRNA silencing results showed that when the dsRNA was present expression of these six genes was significantly decreased (P < 0.01) (Figure 6C).

To confirm the protective effects of these six genes, S. paramamosain were injected intramuscularly with specific dsRNAs before WSSV or V. alginolyticus challenge. There were 16 crabs in each group and experiments were conducted for 144 hours. The control group was injected with PBS solution followed by WSSV challenge. Each point represents the means of a triplicate assays within the SD. Then we tested the mortality rates of crabs when they were infected by WSSV or V. alginolyticus. The data showed that the knockdown of these six genes led to a faster morbidity rate in the WSSV-infected crabs and significantly (P < 0.01) increased the mortality of crabs compared with that of the control WSSV group (Figure 7A). In the first 84 h, knockdown of these six genes also led to a faster morbidity rate in the V. alginolyticus-infected crabs but did not change the mortality rate significantly when compared with that of the V. alginolyticus control group (Figure 7B). However, four genes did show a higher mortality rate than that of the control after they were knocked down.

In this study, we identified 1,799 proteins through the iTRAQ analysis of the S. paramamosain hemocytes proteome and showed that proteins with a mass between 10–60 kDa occupied 59% of all proteins in these animals. The major pathways included metabolism, 226 proteins, translation, 212 proteins, and transport and catabolism, 160 proteins. The important biological processes included cellular and metabolic processes, and the major molecular functions were binding and catalytic activity. No matter for WSSV or V. alginolyticus infection, the organelle and intracellular organelle are the most important, and metabolic pathways were the key pathways. WSSV was found to induce metabolic changes (17), and endocytosis is vital for its replication in crustacean (18). V. alginolyticus infection would damage the mitochondria of hemocytes and then cause hemocyte death in crabs (19). Interestingly, we found that WSSV infection significantly affected more proteins’ expression in host hemocytes than V. alginolyticus infection. Many cytoskeleton proteins contribute to WSSV infection indicating that phagocytosis is the most important cellular process during infection. This finding is accordance with previous reports (11, 20). Interestingly, we found that 106 proteins were differentially expressed simultaneously in WSSV infection and V. alginolyticus infection. These proteins can be categorized into some important pathways including carbon metabolism, ribosome, MAPK signaling pathway, Ras signaling pathway, phagosome, and Hippo signaling pathway. The major molecular functions of these proteins were binding, catalytic activity, and organelle composition.

Then, we chose six differentially expressed genes to investigate their expression and the protective effects against WSSV infection or V. alginolyticus infection. We found that these six genes showed a trend of upregulation at 24 h post-WSSV or V. alginolyticus challenge except for that of myosin-9 in response to WSSV, and only β-actin and sarcoplasmic calcium-binding protein 1 showed the same trend in the proteomic results. We found that the knockdown of these six genes led to an increase in morbidity and mortality in the WSSV-infected crabs when compared with that of WSSV control group. Similarly, knockdown of these six genes also led to an increased morbidity rate in the V. alginolyticus-infected crabs in the first 84 h and four genes showed an increase in the mortality rate when compared to the V. alginolyticus control group. β-Actin and myosin are known to regulate phagocytosis in shrimp and participate in the response to WSSV (11, 20, 21). Phagocytosis is also the key immune response to bacterial challenge in invertebrates (22). In this study, β-actin and myosin-9 were upregulated significantly (P < 0.01) in WSSV infection, but β-actin was downregulated and myosin-9 upregulated in V. alginolyticus infection. When β-actin or myosin-9 was knocked down during WSSV or V. alginolyticus infection, there was an increase in the morbidity and mortality rates. The data indicated that β-actin and myosin-9 play an important role in the immune response to WSSV or V. alginolyticus infection.

Sarcoplasmic calcium-binding protein 1 is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely, to “buffer” cytosolic Ca²⁺, and its expression is downregulated in pre- and postmolt stages compared with intermolt in crayfish (23). Sarcoplasmic calcium-binding protein is also an important allergen in the muscle and shell of Fenneropenaeus merguiensis (24). WSSV infection in Penaeus monodon is facilitated by housekeeping molecules like sarcoplasmic calcium-binding protein 1 (25). When sarcoplasmic calcium-binding protein 1 was knocked down, WSSV infection would cause the animals to die faster and more often, but in V. alginolyticus infection this would only reduce the lifespan of infected crabs but decreased the overall mortality rates. These results revealed that sarcoplasmic calcium-binding protein 1 plays an important role in the immune response to WSSV and plays a different role in the immune response to V. alginolyticus infection. The anti-lipopolysaccharide factors (ALFs) are well known for being the most common effectors affecting the invading microorganisms in crustaceans and its antimicrobial activity in killing Gram-positive and -negative bacteria and viruses (26–31). In this study, ALF 4 and ALF iso4 were upregulated significantly in WSSV and V. alginolyticus infection. When ALF iso4 was knocked down, in either WSSV or V. alginolyticus infection it resulted in a reduced lifespan an increase in the mortality rate. However, the knockdown of ALF 4 leads to increased morbidity and mortality in WSSV-infected crabs and increased morbidity but decreased mortality in V. alginolyticus-infected crabs. The transketolase is required for cancer growth because of its ability to affect the production of NAPDH to counteract oxidative stress (32). The transketolase-like protein 2-like isoform 1 was downregulated significantly in WSSV infection and V. alginolyticus infection. The knockdown of transketolase-like protein 2-like isoform 1 caused an increase in the morbidity and mortality rates following infection with either pathogen.

In this investigation, we reported on the proteome of the mud crab S. paramamosain in response to WSSV or V. alginolyticus infection. We investigated the molecular mechanisms underlying the immune response to WSSV infection in crustaceans. These findings will contribute to our understanding of the molecular mechanisms of immune response to WSSV or V. alginolyticus infection in crab hemocytes.

The animal subjects used in the present study are crab, which are invertebrates and are exempt from this requirement.

FZ and BS designed experiments, analyzed experimental results, and wrote the manuscript; BS, ZhW, ZiW, and XM carried out experiments.

There are no patents, products in development, or marketed products to declare. This does not alter our adherence to all the gene policies on sharing data and materials.