Introduction

The Chinese shrimp, Fenneropenaeus chinensis (F. chinensis), is one of the most commercially important cultured shrimp in China. However, the worldwide outbreaks of viral and bacterial diseases have caused the menaced penaeid shrimp aquaculture a big economic loss. Among all known pathogens of shrimp, the Vibrio has been implicated as the main bacterial pathogen (Baticados et al. 1990 ; Bachère 2000), and white spot syndrome virus (WSSV) is the most popular viral pathogen (Lightner 1996; Yang et al. 2001).

It's well-known that shrimp adopt innate immune system to prevent them from pathogen invasion since lacking adaptive immune systems (Bachère et al. 2004). In the recent years, more studies about crustacean immunology have been reported (Li and Xiang 2013a). Many major effectors of the crustacean immune system, including the pattern-recognition proteins (Cheng et al. 2005; Liu et al. 2007), prophenoloxidase system (Liu et al. 2006), antimicrobial peptides (Destoumieux et al. 2000; Liu et al. 2005), and signaling pathways regulating innate immune responses (Flegel and Sritunyalucksana 2011; Li and Xiang 2013b; Wen et al. 2013), were studied using biochemical and molecular biological approaches. In recent years, the bioinformatic data of shrimp are accumulating at dramatic rate (Zhang et al. 2010b; Leu et al. 2011; Andriantahina et al. 2013), which enable researchers to perform some high-throughput analysis to rapidly screen a plenty of important genes or proteins involved in shrimp immune response, without knowing the detailed molecular mechanisms of immune responses. In transcriptional level, expressed sequence tags analyzing, suppression subtractive hybridization, differential display PCR, and cDNA microarray have been applied to isolate differentially expressed genes of shrimp in response to immune stimulation (Somboonwiwat et al. 2006; Wang et al. 2006; Wang et al. 2008; Robalino et al. 2009; Aoki et al. 2011; Li et al. 2013); the information of mRNA expression level reflected the expression potential of the corresponding proteins. To comprehensively study the gene expression pattern, it is necessary to collect information of protein expression profiles, as proteins take center stage in directing the work of living cell. As a high-throughput characterization method in translational level, proteomics was utilized to identify differentially expressed proteins of shrimp in response to pathogen challenge (Chongsatja et al. 2007; Wang et al. 2007; Rattanarojpong et al. 2007; Chai et al. 2010; Zhang et al. 2010a; Chaikeeratisak et al. 2012).

To develop control strategies to prevent different infectious diseases of shrimp, it is necessary to study the response of shrimp toward different types of pathogen, especially the similar and differential molecular reactions of the shrimp immune system toward different pathogens. The differentially expressed gene in response to WSSV or Vibrio infection within the same batch of shrimp have been screened and compared by cDNA microarray (Wang et al. 2008). However, there is no report on comparing the protein expression patterns of shrimp stimulated by different pathogens. In this study, differentially expressed proteins in the hepatopancreas, a main immune organ in shrimp, were investigated through proteomic approach when shrimp were challenged by WSSV and Vibrio, aiming to compare protein expression patterns in shrimp response to different pathogens.

Materials and Methods

Shrimp and Pathogens Challenge

Healthy Chinese shrimp with an average length of 9.4–10.6 cm were obtained from a local shrimp farm near Qingdao. They were cultured in air pumped circulating seawater at 21 °C with 32 ‰ salinity at least 10 days prior to the experiment. Artificial diet was given two times per day. Shrimp were randomly selected and divided into Vibrio-challenged, WSSV-challenged, and control groups.

In the Vibrio challenge experiment, shrimp were injected with 20 μL heat-inactivated Vibrio anguillarum suspended in normal saline (107 cells/mL). In the WSSV challenge experiment, shrimp were injected with 20 μL tissue homogenate isolated from WSSV-infected shrimp. To prepare the tissue homogenate, 10-g tissues of WSSV-infected shrimp were homogenized in 10 mL PBS-His on ice (Huang et al. 1999). The homogenized tissue was centrifuged at 3,800×g at 4 °C for 15 min. The supernatant was transferred to a fresh tube, sucrose was added to 30 % (w/v) and then the mixture was centrifuged at 38,000×g at 4 °C for 50 min. The pellets were resuspended in 25 mL of normal saline for injection. The control shrimp were injected with 20 μL normal saline. Twelve shrimp from each group were taken out randomly after 24 h. The hepatopancreas were dissected out for further proteomic analysis.

Extraction of Hepatopancreas Proteins

The total protein of shrimp hepatopancreas from Vibrio-challenged, WSSV-challenged, and control groups were extracted as previously described (Jiang et al. 2009). Protein concentration was measured using the Quick Start Bradford Protein Assay Kit 1 (Bio-Rad, Hercules, USA).

2-DE

A pool of hepatopancreas samples isolated from four shrimps (within the same group) was used for resolving the hepatopancreas proteins in a two-dimensional electrophoresis (2-DE) gel. Three independent pools per group (totally twelve shrimps per group) were used for 2-DE analysis. A Bio-Rad Protean IEF Cell System (Bio-Rad, Hercules, USA) was used for the isoelectric focusing (IEF) dimension. Approximately 300 and 600 μg of total protein was loaded onto each 18 cm immobilized pH gradient (IPG) strip pH 4–7 and pH 3–10NL (GE Healthcare, Sweden) separately. The IEF conditions and the following equilibration were as described in the previous study (Jiang et al. 2009). The second dimension electrophoresis was carried out at 14 °C on a Bio-Rad Protean II xi Cell System (Bio-Rad, Hercules, USA). Fourteen percent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 0.165 T, 3 %C tricine-SDS-PAGE were used for the pH 4–7 and pH 3–10NL IPG strips separately. Then the gel was stained with CBB G-250 colloid staining method.

Image Analysis

ImageMaster 2D Platinum 6.0 (GeneBio, Geneva, Switzerland) was used for analyzing 2-DE images including matching spots, quantifying spots, and identifying differences of spot intensity. The spot intensity was quantified by total spot volume normalization and comparison was according to the spot volume percentage. Spots with significant and reproducible changes were considered to be differentially expressed proteins.

In-gel Protein Digestion

The differentially expressed protein spots were manually excised from the gels using 200 μL tips and grinded into small pieces. The following gel destain, dehydration, digestion, and peptides collection procedures were operated as previous described (Jiang et al. 2009).

LC-ESI-MS/MS

Twenty microliters of peptide samples were loaded onto a C-18 column, 100 × 0.18 mm, 5 μm (Thermo Electron Corporation, Waltham, USA) and separated using a gradient of 5 % A and 95 % B for 15 min, 65 % A and 35 % B for 45 min, and 95 % A and 5 % B for 25 min, where A was acetonitrile with 0.1 % formic acid and B was water with 0.1 % formic acid. The C-18 column was connected directly to the LCQ DECA XPplus ion trap mass spectrometer (ThermoQuest, San Jose, USA). Full-scan spectra were recorded in positive mode over the mass range of 300–1,500 Da. The MS/MS data were automatically acquired on the three most intense precursor ions in each full-scan spectrum.

Database Searching

The MS/MS data were firstly searched against the NCBInr and expressed sequence tag (EST) databases using the Mascot online (http://www.matrixscience.com). The searching parameters were set as previously described. Identifications with individual ions scores indicating identity or extensive homology (p < 0.05) were defined positive.

Then the MS/MS spectra were searched again using SEQUEST in the BioWorks 3.1 software package against F. chinensis peptides database (FcDB). The construction of FcDB and searching parameters were as previously described (Jiang et al. 2009). Those with Xcorr score equal to or above 1.5, 2.2, or 3.5 for singly, doubly, or triply charged precursor ions, respectively, and Delta Cn above 0.1 was defined as positive identifications.

Results

Comparison of Protein Expression Profiles of Hepatopancreas Between Vibrio-Challenged Shrimp, WSSV-Challenged Shrimp, and Normal Shrimp

As shrimp hepatopancreas protein mainly ranged from 15,000 to 90,000 Da, pH 4–7 (Jiang et al. 2009), we first run pH 4–7 strips and 14 % SDS-PAGE gels to decrease the disturbance of high abundance proteins in acidic area and enhance the resolution of 2-DE gels (Fig. 1a–c). Approximately 680–710 spots on each gel were distinguished by ImageMaster 2D. We also hope to get a complete view of the shrimp hepatopancreas protein ranging from pI acidic to basic, molecular weight low to high, especially low molecular weight proteins that might contain antibacterial peptides. So we run a parallel gel of pH 3–10NL strip and 0.165 T, 3 %C tricine-SDS-PAGE (Fig. 1d–f). Approximately 760–800 spots on each gel were distinguished.

Fig. 1
figure 1

2-DE maps of Chinese shrimp hepatopancreas. a IPG 4–7, SDS-PAGE; and control, b IPG 4–7, SDS-PAGE, and heat-inactivated Vibrio anguillarum-challenged group; c IPG 4–7, SDS-PAGE, and WSSV-challenged group; d IPG 3–10NL, tricine-SDS-PAGE, and control; e IPG 3–10NL, tricine-SDS-PAGE, and heat-inactivated Vibrio anguillarum-challenged group; and f IPG 3–10NL, tricine-SDS-PAGE, and WSSV-challenged group

Full size image

Using ImageMaster 2D software to analyze the total 18 2-DE gels, 53 protein spots showed significantly differential expression in the hepatopancreas of Vibrio-challenged shrimp compared with control shrimp (Fig. 1), including 8 upregulated (expression levels were ≥1.5-fold of controls) spots and 45 downregulated (expression levels were ≤0.67-fold of controls) spots; 52 protein spots showed significantly differential expression in the hepatopancreas of WSSV-challenged shrimp compared with control shrimp (Fig. 1), including 11 upregulated (expression levels were ≥1.5-fold of controls) spots and 41 downregulated (expression levels were ≤0.67-fold of controls) spots.

Protein Identification

The differential expression proteins were analyzed by LC-ESI-MS/MS. By combining results of searching MS/MS spectra data against NCBInr and EST database by Mascot and against FcDB by SEQUEST, more abundant and accurate peptide identifications were achieved (Porubleva et al. 2001; Kim et al. 2004). In hepatopancreas of Chinese shrimp challenged with heat-inactivated Vibrio anguillarum, 48 of the total 53 differentially expressed protein spots including 6 upregulated (representing 5 proteins) and 42 downregulated (representing 31 proteins) were successfully identified, including 5 protein spots related to energy production, 5 protein spots related to immune, 224 protein spots involved in the metabolism, 4 antioxidant protein spots, 7 chaperone spots, 1 translation related protein spot, and 2 ungrouped protein spots; in hepatopancreas of Chinese shrimp challenged with WSSV, 49 differentially expressed protein spots including 11 upregulated (representing 9 proteins) and 38 downregulated (representing 27 proteins) were successfully identified, including 7 protein spots related to energy production, 9 protein spots related to immune, 21 protein spots involved in the metabolism, 5 antioxidant protein spots, and 7 chaperone spots. Thirty-one of these identified altered expressed protein spots were found in both Vibrio-challenged and WSSV-challenged groups, and most of them had the similar alteration trend response to Vibrio and WSSV challenge. Seventeen or 18 of these identified altered expressed protein spots were found only in Vibrio-challenged group or WSSV-challenged group (shown in Table 1). Some different spots were identified as the same proteins, and this might be the results of different protein isoforms or posttranslational modifications.

Table 1 Differentially expressed proteins identified by LC-ESI-MS/MS in hepatopancreas of Chinese shrimp challenged by Vibrio and WSSV
Full size table

Discussion

The aim of this study was to investigate the protein expression changes in the hepatopancreas of Chinese shrimp challenged with two different pathogens and get a better understanding of shrimp response to immune stimulation. Here we successfully identified 48 and 49 spots in Vibrio and WSSV-challenged group, respectively, and most of them have important biological functions and should play important roles in immune response of Chinese shrimp.

Proteins Related to Energy Production

In this study, we observed the expression level of some proteins related to energy production changed after pathogen challenge, and some of them had different alteration trend between Vibrio and WSSV-challenged group. Alpha-amylase, the most important glucosidase in shrimp, catalyzes the hydrolysis of internal alpha-1,4-glucan links in polysaccharides, yielding a mixture of maltose and glucose, the fermentable substrate (Wormhoudt and Sellos 1996). In this study, we observed one spot (v9) identified as preamylase 1, the precursor of alpha-amylase, showed downregulation after Vibrio challenge, which would lead to a decrease in ATP production due to decreased fermentable substrate. In WSSV-challenged group, the same spot (w10) showed upregulation, and another spot (w9) also identified as preamylase 1 showed downregulation. Meanwhile, we observed that two key glycolytic enzymes, phosphopyruvate hydratase and triosephosphate isomerase showed downregulation after Vibrio challenge (v25 and v17). In WSSV-challenged group, only w14 (same spot as v17) was downregulated, while w18 (same spot as v25) and w45 were upregulated. This may reflect that after Vibrio challenge, the energy production was depressed, while after WSSV challenge, the energy production was maintained in shrimp. We also observed that two spots identified as arginine kinase were downregulated in both Vibrio- and WSSV-challenged groups. Arginine kinase catalyzes the reversible transfer of high-energy phosphate from arginine phosphate to ADP for regenerating ATP when acute lack of energy occurs in invertebrate (Yao et al. 2005). The downregulation of arginine kinase might be a result of the decreased concentration of arginine phosphate and ATP due to acute energy consumption. This observation indicated that shrimp confront severe energy shortage after Vibrio or WSSV challenge.

Based on the results above, we concluded after Vibrio challenge, the energy production in shrimp reduced as a result of glycolysis depression, and after WSSV challenge, energy production was at least maintained as before. However, both Vibrio- and WSSV-challenged shrimp confronted energy shortage, so as the energy reservoir, arginine phosphate were consumed. It seems probable that this different energy metabolism strategy between Vibrio- and WSSV-challenged shrimp is because WSSV replication requires more energy in WSSV-challenged group.

Proteins Related to Immune

In our study, we also observed the expression of several immune-related proteins varied after pathogen challenge. Chitinase, an enzyme involved in digestion of chitin-containing food in crustacean hepatopancreas, may play an important role in innate immunity. In Crassostrea gigas, chitinase mRNA was upregulated in hemocytes after bacterial challenge (Badariottia et al. 2007). In the stomach of WSSV-infected L. vannamei, chitinase also showed upregulation (Wang et al. 2007). In this study, one spot in Vibrio-challenged group and two spots in WSSV-challenged group identified as chitinase also showed upregulation. Macrophage migration inhibitory factor, initially identified as a T cell cytokine, plays an important role in the innate immune response. It was reported that macrophage migration inhibitory factor-deficient macrophages were hyporesponsive to lipopolysaccharide and gram-negative bacteria (Nishihira 2000). In this study, one macrophage migration inhibitory factor spot was downregulated in both Vibrio- and WSSV-challenged shrimp which might reflect that the phagocytosis in shrimp was depressed after pathogen challenge.

The expressions of four proteases, including chymotrypsin BI, trypsin, carboxypeptidase, and zinc proteinase Mpc1 varied in WSSV-challenged shrimp but did not vary in Vibrio-challenged shrimp. Chymotrypsin BI and trypsin, the major proteinase component in shrimp hepatopancreas, not only are involved in digestion but also participate in many aspects of invertebrate immunity (Gorman and Paskewitz 2001). In insects, chymotrypsin-like serine protease has been found related to immune defense reactions against bacteria, yeast, and baculovirus (Finnerty et al. 1999; Guedes et al. 2005), and in F.chinensis, chymotrypsin-like serine protease showed upregulation after pathogen infection (Shi et al. 2008). In crustaceans, trypsin can significantly trigger the activation of prophenoloxidase, whose active form, phenoloxidase, catalyzes the early steps in the pathway to melanin formation in innate immunity and is also responsible for pigmentation (Söderhäll and Cerenius 1998; Zufelato et al. 2000; Lai et al. 2005; Thomas-Guyon et al. 2009). In most cells of a mammalian, carboxypeptidase participates in the formation of protease–proteoglycans complex, an important component in innate immune system of a mammalian (Stevens and Adachi 2007). In WSSV-challenged group, these three proteins were all downregulated, and this might reflect that the immune system in shrimp was defected after WSSV challenge. A similar result was also observed in the stomach of L. vannamei challenged with WSSV (Wang et al. 2007). Zinc proteinase Mpc1 belongs to metalloendoprotease and participates in many biological processes, including the rebuilding of connective tissue and signal peptide removal (Dumermuth and Sterchi 1991). It was reported that in L. vannamei and Marsupenaeus japonicus, the transcriptional level of zinc proteinase Mpc1 in WSSV-resistant individuals was higher than the susceptible ones (Pan et al. 2005; Zhao et al. 2007). In this study, we observed three altered expressed spots identified as zinc proteinase Mpc1 in WSSV-infected shrimp. Two of them showed upregulation (w26, w27), and one of them showed downregulation (w4), reflecting that zinc proteinases with different posttranslational modifications had different functions in anti-WSSV defense response. Neprilysin is also a zinc metalloendopeptidase, involved in the metabolism of many regulatory peptides in the mammalian nervous, cardiovascular, inflammatory, and immune systems (Turner et al. 2001). For invertebrate, the putative zinc-dependent protease homologous to mammalian neprilysin was proved to be crucial to the immune response in Manduca sexta (Willott and Tran 2002). In this study, neprilysin was upregulated after Vibrio stimulation, reflecting that the zinc proteinases may be also important in anti-Vibrio defense response.

Cathepsin L is a kind of cysteine proteases of papain family, stored in lysosomes in the form of proenzyme. It participates in a number of biological processes, such as prohormone activation, antigen presentation, and the development of tissues or organs. In shrimp, the main function of cathepsin L is digestion (Hu and Leung 2007). It was reported that the expression of cathepsin L was downregulated in WSSV-infected Penaeus monodon in transcriptional level (Pongsomboon et al. 2008). In this study, we found that cathepsin L was downregulated in Vibrio-challenged Chinese shrimp but did not change in WSSV-challenged group. We also found that a signal transduction protein related to immune was downregulated in the hepatopancreas of Vibrio-challenged Chinese shrimp. This protein, containing a TBC1 domain, is a Rab-like GTPase. Rab GTPases play important roles in regulating the trafficking of membrane including phagosome formation, phagosome movement, and membrane fusion (Rak et al. 2000). It was reported recently that in Marsupeneaus japonica, phagocytic percentage was significantly decreased when the Rab gene was silenced by RNAi, and the overexpression of Rab gene led to the increase of phagocytic percentage, suggesting that Rab was essential in the regulation of hemocytic phagocytosis of shrimp (Zong et al. 2008). The downregulation of Rab GTPase in this study might reflect phagocytosis of Chinese shrimp decreased after Vibrio challenge, leading to weakened immunity of Chinese shrimp.

The expression pattern of immune-related proteins after pathogen challenge supports very important information to explore the immune response mechanism in Chinese shrimp. From the abovementioned, we could see that there were few in common of the differentially expressed proteins related to immune function between shrimps challenged with Vibrio and WSSV. This was a persuasive evidence that the shrimp adapted very different strategies toward different pathogen infection, which was observed in the study of screening differentially expressed genes at the mRNA level in Chinese shrimp challenged with WSSV and Vibrio (Wang et al. 2008).

Proteins Involved in the Metabolism

According to the expression profiles of differently expressed proteins related to energy production in shrimp after Vibrio and WSSV challenge that, we can see after Vibrio or WSSV challenge, shrimp suffer the energy shortage. In order to maintain ATP demand and supply balance, shrimp were inclined to suppress energy-consuming processes such as reducing metabolic rate. In our present study, a number of enzymes participated in the metabolism of nucleoside, lipid, and amino acid, and one-carbon unit showed differential expression after pathogen challenge, and most of them were downregulated.

Adenosine kinase, which catalyzes the phosphorylation of adenosine to AMP, is a key enzyme in purine salvage pathway (Decking et al. 1997). Transketolase, catalyzing transfer of a two-carbon fragment from a ketose to an aldose in pentose phosphate pathways, has a controlling role in the supply of ribose units for nucleoside biosynthesis (Turner 2000). Phosphoribosylaminoimidazole carboxylase is an enzyme involved in nucleotide synthesis. It catalyzes the conversion of 5′-phosphoribosyl-5-aminoimidazole into 5-phosphoribosyl-4-carboxy-5-aminoimidazole (Meyer et al. 1999). These three enzymes which participated in the metabolism of nucleoside were all found differentially expressed in Chinese shrimp challenged with Vibrio and WSSV. Three spots were identified as adenosine kinase in Vibrio-challenged group with two upregulated (v38, v12) and one downregulated (v11). There were also three spots identified as adenosine kinase in WSSV-challenged group. The spot w36 (same spot as v38) showed upregulation, while w12 (same spot as v11) and w13 showed downregulation. Two spots (v16, v39) identified as transketolase showed downregulation in Vibrio-challenged group, while v30 were upregulated. In WSSV-challenged group, w38 (same spot as v39) also showed downregulation, but w29 (same spot as v30) showed upregulation. Phosphoribosylaminoimidazole carboxylase was found upregulated only in WSSV-challenged group. To sum up, the enzymes related to nucleoside metabolism tended to be downregulated in Vibrio-challenged group, reflecting a suppression of nucleoside biosynthesis took place to save energy; while the upregulation of these enzymes in WSSV-challenged group might be a result of the virus duplication.

Cystathionine beta-lyase catalyzes the alpha, beta-elimination of cystathionine to produce homocysteine, pyruvate, and NH3, and is a key enzyme in methionine biosynthesis (Ravanel et al. 1996). Pyrroline-5-carboxylate dehydrogenase oxidizes pyrroline-5-carboxylate which is a physiological intermediate in the interconversions of proline, ornithine, glutamate, and arginine, to glutamate (Mohamed et al. 2008). These two enzymes involved in amino acid metabolism showed obvious downregulation in both Vibrio- and WSSV-challenged group which might reflect depressions of some amino acid-related metabolism pathways. Pyruvate, an intermediate of several metabolic pathways, can convert to alanine and take part in amino acid metabolism (Cody et al. 2000). Hydroxyacylglutathione hydrolase participates in pyruvate metabolism, with the common substrate methylglyoxal, the product is d-lactate (Vander 1993). Aspartate aminotransferase, also known as glutamic oxaloacetic transaminase, catalyzes the reversible reaction of aspartate and 2-oxoglutarate yielding glutamate and oxaloacetate (Kuramitsu et al. 1985). These four enzymes showed downregulation in Vibrio-challenged group, and three of them except hydroxyacylglutathione hydrolase were downregulated in WSSV-challenged group. This reflected that some amino acid-related metabolism pathways were depressed to save energy after pathogen infection.

One-carbon metabolism can supply organisms to the one-carbon units, which has important biological significant in synthesizing proteins, nucleic acids, pantothenate, and many methylated molecules (Bailey and Gregory 1999). Here, we identified five enzymes related to one-carbon metabolism. Homocysteine S-methyltransferase catalyzes the S-adenosylmethionine, a universal methyl donor of numerous methylation reactions and l-homocysteine to produce S-adenosyl-l-homocysteine and l-methionine, resulting to the one-carbon unit transferring. Formyltetrahydrofolate dehydrogenase participates in folate metabolism, and its downregulation would decrease the concentration of S-adenosylmethionine (Lu 2000; Anguera et al. 2006). S-Adenosyl-l-homocysteine hydrolase catalyzes the reversible hydration of S-adenosyl-l-homocysteine, a potent inhibitor of all methylation reactions, to adenosine and homocysteine. In eukaryotic cells, S-Adenosyl-l-homocysteine hydrolase is believed to activate methyl cycle by inhibiting S-adenosyl-l-homocysteine (Shu et al. 2006). Glutamate carboxypeptidase is a folate hydrolase, playing an important role in folate metabolism (Rawlings and Barrett 1997). Formylglutathione hydrolase is a thioesterases involved in detoxification of formaldehyde which is synthesized from choline oxidation (Hanson et al. 2000). In our study, except glutamate carboxypeptidase that was observed downregulated in Vibrio-challenged group, other four enzymes related to one-carbon metabolism showed downregulation in both Vibrio- and WSSV-challenged groups. We could conclude that some one-carbon units involved in metabolisms were suppressed in the hepatopancreas of Chinese shrimp after pathogen infection, which lead to a suppression of other metabolic pathway.

Two proteins involved in carbohydrate metabolism and one-lipid metabolism-related enzyme also showed downregulation in shrimp after pathogen infection. Fructose-1,6-biphosphatase converts fructose-1,6-bisphosphate to fructose 6-phosphate in gluconeogenesis, which is an energy-consuming process, and is suppressed when energy deficits happen. This enzyme was downregulated in both Vibrio- and WSSV-challenged groups. Beta-galactosidase, catalyzes the hydrolysis of beta-galactosides into glucose and galactose, showed downregulation in WSSV-challenged group. 3-hydroxyacyl-CoA dehydrogenase is an oxidoreductase that participates in the fatty acid metabolism, and this enzyme was downregulated in Vibrio-challenged group.

Aldehyde dehydrogenase catalyzes the conversion of aldehydes to its corresponding acids by means of an NAD(P)+-dependent irreversible reaction. It was reported that aldehyde dehydrogenase was expressed at basal level under hypoxic conditions in fungus (Shimizu et al. 2009). In this study, we also observed two kinds of aldehyde dehydrogenase that showed downregulation in Vibrio- or WSSV-challenged groups (v20 and w33), which might be a result of the reducing environment in hepatopancreas of shrimp after pathogen infection.

Antioxidant Proteins

Phagocytosis is an important immune defense reaction in shrimp hemocytes (Lee and Söderhäll 2002). During this course, the host enhances the production of reactive oxygen species (ROS), known as respiratory burst, to kill foreign invaders efficiently (Bogdan et al. 2000). However, the excessive ROS may cause serious damage to cellular macromolecules, such as nucleic acids, lipids, and proteins (Halliwell and Gutteridge 1999). To maintain the proper functions of cells, excessive ROS should be eliminated, which is performed by antioxidants. In this study, we observed three antioxidant proteins, mitochondrial manganese superoxide dismutase, fatty acid-binding protein 10, and carbonyl reductase (NADPH) 1, that were downregulated in both Vibrio- and WSSV-challenged groups. Mitochondrial manganese superoxide dismutase catalyzes the dismutation of two molecules of superoxide anion into water and hydrogen peroxide (Zhang et al. 2007). Fatty acid-binding protein 10 can bind long-chain fatty acid oxidative products with high affinity and capacity to scavenge them (Rajaraman et al. 2007). Carbonyl reductase 1 detoxificates the lipid peroxidation produced under oxidative conditions (Forrest and Gonzalez 2000). The depressed expression of these three antioxidant proteins in this study might reflect that in the hepatopancreas of Chinese shrimp, it was not an oxidative environment as a result of respiratory burst after pathogen stimulation. It was reported that in the hemocytes of L. vannamei injected with Vibrio alginolyticus, the respiratory burst and superoxide dismutase activity varied at different time points. In the first 24 h after infection, respiratory burst and superoxide dismutase activity decreased as a result of the weakened shrimp's immunity, while a slow recovery of respiratory burst and superoxide dismutase activity happened during 72–96 h postinjection (Li et al. 2008). This might explain why in some reports antioxidant protein expression was provoked by pathogen stimulation (Chai et al. 2010).

Chaperones and Proteins Related to Translation

We found three differentially expressed chaperone proteins in hepatopancreas of shrimp after pathogen infection. Protein disulfide isomerase (PDI) catalyzes the native disulfide bonds formation and rearrangement and participates in protein folding, assembly, and posttranslational modification (Freedman et al. 1994; Tu and Weissman 2004). In the present study, we identified five spots as PDI in Vibrio-challenged group, including one upregulated and four downregulated spots. The corresponding spots in WSSV-challenged group showed the same alteration trend as that in Vibrio-challenged group. The complex PDI expression pattern might reflect that the changes in posttranslational modifications of PDI played important roles in immune response, which should be further studied. Heat shock protein 70, a kind of ubiquitously expressed molecular chaperone, assists the folding of newly synthesized peptides in the cell and helps to protect cells from stress (Mayer and Bukau 2005). Proteasomes are very large barrel-like protein complexes, and beta subunits are components of the core structure. The main function of the proteasome is to degrade unneeded or damaged proteins into peptides by proteolysis, a chemical reaction that breaks peptide bond. These peptides are further degraded into amino acid molecules for synthesizing new proteins (Peters et al. 1994). Ribosomal protein L5 is a component of 50S subunit in ribosome, a molecular machine for translation in cells. In this study, heat shock protein 70 and proteasome beta subunit showed downregulation in both Vibrio- and WSSV-challenged Chinese shrimp, and ribosomal protein L5 was downregulated in Vibrio-challenged group. Taking together, the PDI showed downregulated; we conclude that protein synthesis was greatly suppressed in response to pathogens stimulation as a result of energy shortage. This result is also in agreement with the heat shock protein 70 expression pattern in epithelium of Penaeus monodon in response to WSSV infection (Wu et al. 2007).

Ungrouped Proteins

Oncoprotein nm23 is a nucleoside diphosphate kinase, which plays roles in maintaining a pool of nucleoside triphosphate required for biosynthesis. It was reported that overexpression of nucleoside diphosphate kinase could protect cell from excessive production of ROS under oxidative stress (Benhar et al. 2002). We also found that in shrimp under hypoxic condition, oncoprotein nm23 was downregulated at protein level (Jiang et al. 2009). In the present study, nm23 was observed downregulated after Vibrio infection, and this might relate to the suppression of ROS in response of Vibrio stimulation deduced in this study. Heat-responsive protein 12 had significant similarity to DnaK/heat shock protein 70, expressed highly in mouse liver and kidney and was upregulated after heat shock (Samuel et al. 1997). In this study, it was downregulated after Vibrio stimulation, but the significance of this phenomenon is unknown.

From the protein expression patterns of Chinese shrimp in this study, we found that the mechanisms of Chinese shrimp in response to Vibrio and WSSV challenge had something in common and also had their own characteristics. Many proteins participated in the interaction between host and pathogens; their different expression patterns between shrimps challenged with Vibrio and WSSV indicated that host adapted different strategies to resist different pathogens. This study set basis for dissecting the immune mechanisms in shrimp and provided important information to develop anti-disease strategy, aiming at different pathogens infection in shrimp culture. Some results in this study was not absolutely in agreement with the existing studies; this might be because of the differences in shrimp species, organs, pathogens, and the immune stage of the shrimp (Wu et al. 2007; Qiao et al. 2011). Further studies on shrimp immune response in other organs and at other time points should be performed. We found some spots identified as one protein that showed opposite alteration trends after pathogen challenge; this might be because they were same proteins with different post translational modifications that played different roles in immune response. The function of these proteins should be focused in the future studies. We used the inactivated but not live Vibrio to challenge shrimp in this study, so the host's responses were caused by pattern-recognition molecules, without the influences of live pathogen to host. Future efforts would also be focused on the protein expression profiles of shrimp in response to other kinds of pattern-recognition molecules and live pathogens.