Introduction

The worldwide costs of disease control in the crustacean aquaculture industry are widely acknowledged [1–3]. To reduce the occurrence of disease in crustacean aquaculture, great efforts have been done to study the innate immune system of crustacean. According to previous studies, the primary immune response in crustacean is the melanization of pathogens and damaged tissues [4, 5]. This important process is controlled by the ProPO-activation pathway [4–6], which is a phenoloxidation cascade comprising of pattern recognition proteins, several serine proteases, their inhibitors and terminates with the zymogens, ProPO [4]. The ProPO activating enzyme or factor are serine proteases, which cleave ProPO to generate the active enzyme, phenoloxidase. Activation of ProPO is mediated by a proteinase cascade plus additional factors. The terminal serine proteinase that carries out the proteolysis of the ProPO precursor has been variously named the ProPO activating factor (PPA) [4].

Several arthropod haemocytes derived cell lines dispaly ProPO mRNA expression capability, and ProPO expression may also be used as a marker to follow haemocyte maturation; in Drosophila melanogaster, ProPO expression accompanies crystal cell maturation. Both cellular differentiation and ProPO expression can be annulled by interfering with gene coding for the transcription factors [7]. In Pacifastacus leniusculus, immature haemocytes within the hematopoietic organ do not express the ProPO, whereas mature haemocytes in circulation do [6]. It seems that ProPO expression level contains the information about ProPO function at different life phases [8]. ProPO are so important enzymes in crustaceans that they have been cloned from several species, including Pacific white shrimp Litopenaeus vannamei (GenBank accession number EU373096 and AY723296 and EF565469 and EF115296), black tiger shrimp Penaeus monodon (AF099741.1, AF521948.1). Recently, the characterization of ProPO and its role in immunomodulation in white shrimp (L. vannamei) and giant freshwater prawn (Macrobrachium rosenbergii) were reported [9, 10].

Several immune parameters of the crustacean, including haemocyte counts, PO activity, respiratory bursts (production of superoxideanion), phagocytic activity and clearance efficiency, and the susceptibility to pathogens in relation with the molt cycle have been reported in crustaceans [11–13].The transcripts level change of ProPO in haemocytes and hepatopancreas after V. anguillarum injection has not been investigated while V. anguillarum was a significant pathogenic bacteria [14].

Materials and methods

Experimental shrimp and immune challenge

A batch of apparently healthy shrimp F. chinensis, with body length of 10.5 ± 0.5 cm were purchased from a local shrimp farm. They were acclimated in seawater for 7 days in the lab. Hemolymph was collected from the ventral sinus located at the first abdominal segment with an equal volume of anticoagulant-modified Alservier solution (27 mM sodium citrate, 336 mM NaCl, 115 mM glucose, 9 mM EDTA, pH 7) [15]. Haemocytes were isolated by centrifugation at 800g, at 4°C for 10 minutes and immediately preserved in liquid nitrogen. Different shrimp tissues were dissected out at the same time, and preserved in liquid nitrogen for RNA extraction.

Challenged experiments were performed by injecting 20 μl suspension of inactivated V. anguillarum (108 CFU/ml) in physiological saline solution into the last abdominal segment of each shrimp. Shrimp injected with 20 μl of sterile physiological saline were maintained as controls. Haemocytes, hepatopancreas and lymphoid organ from six experimental and six control shrimps were collected at 6, 12, 24, and 72 h post injection (hpi) and preserved for real time reverse transcript polymerase chain reaction (RT-PCR).

Total RNA extraction and the cDNA synthesis

Total RNA of each sample was extracted using Unizol reagent (Boxing Co. Limited., Shanghai, P. R. China). RNA quality was assessed by electrophoresis on 1% agrose gel. Total RNA was treated with RQ1 RNase-Free DNase (Promega) to remove contaminated DNA. cDNA was synthesized from 5 μg total RNA by M-MLV reverse transcriptase (TaKaRa, Dalian, China) following the manufacturer’s protocol with Hexadeoxyribonucleotide Mixture primer [16].

Cloning and sequencing of ProPO cDNA fragment

A pair of primers, ProPO f1 and ProPO rl, was designed according to the EST from haemocytes cDNA library. PCR amplification was performed using the template cDNA from haemocytes (one cycle of 94°C for 5 min; 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 40 s; followed by one cycle of 72°C for 10 min). PCR products were isolated on 3% agarose gels, purified by PCR purification kit (Promega), and cloned into PMD 18-T vector (TaKaRa). The resultant recombinant plasmid was then transformed into TOP 10, and the positive transformants were screened by PCR.

3′ and 5′-RACE

Based on the partial sequence of ProPO, the 3′ and 5′ ends were obtained by the rapid amplification of cDNA ends (RACE) approaches, using gene-specific primers and adapter primers. For ProPO 3′-RACE, nested PCR was applied and the cDNA template used was from haemocyte RNA. PCR reaction was performed with gene-specific outer primer ProPO f2 and AP primer. The PCR was carried out according to the program of 94°C for 5 min, 35 cycles of 94°C for 50 s, 55°C for 50 s and 72°C for 1 min, and then an extension of 72°C for 10 min. Then, the inner primer ProPO f3 and AP primer were used to amplify the 3′ terminal sequence with the same conditions, except for the annealing temperature of 58°C [16].

For ProPO 5′-RACE PCR, nested PCR strategy was employed to increase specificity. The outer gene-specific primer ProPO r2 and the gene-specific inner primer ProPO r3 were designed from the 3′-RACE. The PCR conditions were the same as mentioned above. The specific amplicons obtained were cloned into the TOPO-TA cloning vector (Invitrogen), sequenced, and aligned with the original sequences. The continuity of the 3′- and 5′-RACE products was verified by contiguous overlapping regions. For both ProPO 3′ and 5′-RACE PCR, cDNA template used was from haemocytes RNA.

Sequence analysis of ProPO

The nucleotide and deduced amino acid sequences of ProPO cDNA were analyzed using BLAST algorithm (NCBI, http://www.ncbi.nlm.nih.gov/BLAST/). The signal peptide, extracellular domain, transmembrane, cytoplasmic domains and other characteristic structures of ProPO were predicted by the simple modular architecture research tool (SMART) program (http://smart.embl-heidelberg.de/). Phylogenetic and molecular evolutionary analyses of the predicted amino acid sequences of different crustacean ProPO were conducted using neighbor-joining method with the software of molecular evolution genetics analyses (MEGA) version 3.1 [17].

Real time PCR analysis of gene expression and relative quantitative statistical analysis

Relative quantification of mRNA transcript abundance required a Taqman probe and a pair of primers for Prophenoloxidase (ProPO) and endogenous reference Triosephosphate isomerase (TPI). Primers and probes of TPI gene were the same as reported [18]. Primers and probe sets for ProPO were designed using the software package Primer Express (Applied Biosystems, USA). Each primer and probe set was designed using the default parameters optimized by ABI Prism 7900 HT (Applied Biosystems, USA). The sequences of all the primers and probes used are summarized in Table 1.

Table 1 Oligonucleotide sequences of all used primers
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Before comparing control and stimulated samples, the overall variations of 18S rRNA expression were first examined. Real-time RT-PCR assays were carried out in a fluorometric thermal cycler (ABI PRISM 7900 HT Sequence Detection System, Applied Biosystems, New Jersey, USA) with a final volume of 25 μL. Each sample was run in triplicate and NTC (no template controls) were included for each primer and probe set. Fluorescence was monitored during every PCR cycle at the annealing step. The RT-PCR mixture contained the following: 1× Taqman One-Step RT-PCR Master Mix Reagents (Applied Biosystems, New Jersey, USA), 200 nM primers and 300 nM probes. Real-time RT-PCR were run with the following program: 30 min at 48°C, 10 min at 95°C, 45 cycles of 15 s at 95°C, 30 s at 55°C, 30 s at 72°C. All the primers and fluorescent probes were synthesized and purified by Shanjing Co. Ltd (Shanghai, China).

2−ΔΔCt method was used to analyze the quantitative expression after verifying the amplification efficiency of endogenous and target genes. The amplification efficiency of each gene was calculated from the slope of a line generated by plotting the Ct value against the log10 of the dilution factor. No significant difference was found for the ProPO and TPI gene (t-test, P > 0.05). A plot of the log cDNA dilution versus ΔCtTPI and ΔCt ProPO was made to compare their amplification efficiency. The slope of TPI was −3.4206, and ProPO was −3.4952, respectively. The absolute value of their dispersion was less than 0.1. Further, the amplification efficiency of TPI and ProPO were 1.9425 and 1.9324 respectively. The two genes’ amplification values were very closed (dispersion between them <0.04), so the assumption seems valid and the ΔΔCt method could be used to analyze the data [19].

Results

Full length cDNA of ProPO

The full-length nucleotide sequence and the deduced amino acids sequence of shown in Fig. 1. The complete sequence was 3040 base pairs and contained an open reading frame (GenBank accession number EU015060) beginning at the nucleotide 74 and ending at the nucleotide 2132. It yielded a deduced polypeptide of 686 amino acids. SignalP software (ExPASy) predicted that the first 12 amino acids form a signal peptide. The protein has a predicted molecular mass of 78,221 Da and a predicted isoelectric point of 5.86. In Fig. 2, the black parts of the alignment were the same amino acids in five shrimps. The deduced amino acid sequence of ProPO showed 93% identity with that of P. monodon, 92% with that of P. semisulcatus, 87% with that of Litopenaeus vannamei, 80% with that of Marsupenaeus japonicus.

Fig. 1
figure 1

Complete nucleotide and deduced amino acid sequence of ProPO from F. chinensis. The letters in box indicated start codon (ATG), stop codon (TGA) and the polyadenylation signal sequence (AATAAA). The putative sequence of signal peptide is shaded by gray. Three Kinase binding sites (V51, V138, V300) are labeled as Ⓥ, one tyrosine kinase (399–423 amino acid) with gray, one NOZZLE site (598–617 amino acid) is framed α2-macroglobulin (GCGWPQHM) are labeled with underline

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Fig. 2
figure 2

Multiple alignment of amino acid sequences of clip domain ProPO homolog: F.chinensis (present study), Penaeus monodon (AAM77689), Penaeus semisulcatus (AAM77690), Litopenaeus vannamei (AAW51360), Marsupenaeus japonicus (BAB83773). Shaded areas indicate complete conservation in these five species. Six conserved histidines are marked with asterisks

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Three Kinase binding sites (V51, V138, V300), one tyrosine kinase (399–423 amino acid), one NOZZLE site (598–617 amino acid) and α2-macroglobulin (GCGWPQHM) were also present in F. Chinensis ProPO (in Fig 1). Multiple alignments with other crustaceans ProPO showed high conservation in six histidines sites (H207, H211, H233, H366, H370 and H408) which marked with asterisks (in Fig. 2), which were putative copper-binding sites.

Phylogenetic analysis of ProPO

To examine the relationships among various prophenoloxidases (ProPOs), a phylogenetic tree was constructed using the overall amino acid sequences of 14 crustacean ProPOs (Fig. 3). Branch 1 consisted of two subgroups. FcProPO together with other shrimp polyphenoloxidases (P. semisulcatus, P. monodon, L. vannamei and M. japonicus) was clustered into the same subgroup. ProPOs from lobsters (H. americanu and H. gammarus) and crayfish (P. clarkia and P. leniusculus) were placed into another subgroup. Branch 2 was composed of ProPOs from two crabs (S. serrata and C. magister).

Fig. 3
figure 3

A phylogenetic tree of 14 crustacean ProPOs. The reliability of each branch was tested by 1,000 bootstrap replications. Numbers at the nodes indicated bootstrap values. Sequences obtained from GenBank include: Fc (EU015060), Ps(AF521949_1), Pm (AF521948_1), Lv (AAW51360.1),Mj (BAB70485.1), Lv2 (ABY81277.1), Ha (AAT73697.1), Hg (CAE46724.1), Pc (ABR12412.1), Pl (CAA58471.1), Ss (ABD90511.1), Cm (ABB59713.1), Mr (ABA60740.1), Af (CAO98768.1). Abbreviations are: Fc, F. chinensis; Ps, Penaeus semisulcatus; Pm, Penaeus monodon; Lv, Litopenaeus vannamei; Mj, Marsupenaeus japonicus; Lv2, Litopenaeus vannamei prophenoloxidase -2; Ha, Homarus americanus; Hg, Homarus gammarus; Pc, Procambarus clarkia; Pl, Pacifastacus leniusculus; Ss, Scylla serrata; Cm, Cancer magister; Mr, Macrobrachium rosenbergii; Af, putative prophenoloxidase of Artemia franciscana

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Expression of ProPO in haemocytes, hepatopancreas and lymphoid organ challenged by V. anguillarum

The results of relative ProPO expression in haemocytes, hepatopancreas and lymphoid organ were also calculated by 2−ΔΔCt method. The information of ProPO expression was given after t testing. Before challenged by V. anguillarum, the highest level of mRNA was obtained in haemocytes and the lowest was got in hepatopancreas (Fig. 4).

Fig. 4
figure 4

Relative expression of ProPO in different tissues of F. chinensis

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After challenged by V. anguillarum, the expression of ProPO in haemocytes, increased significantly (P < 0.05) from 12 h to 72 h in the infection group, while the control group changed little. In lymphoid organ, ProPO expression level was up-regulated to its peak at 6 hpi, then decreased slowly to normal level (P < 0.05) (Fig. 5). There were remarkably significant difference between the control groups and the injection groups at 6 hpi and 12 hpi. In hepatopancreas, ProPO expression in infected group was sharply decreased at 6 hpi (P < 0.05) and showed little up-regulation compared with that of control group at 12 hpi and 24 hpi.

Fig. 5
figure 5

The mRNA level expression of ProPO at different infection time in the three different tissues at 0, 6, 12, 24, 72 hpi. The columns with grids are the infection groups, the blank columns are control groups. (a) Expression of ProPO in haemocytes, (b) Expression of ProPO in lymphoid organ, and (c) Expression of ProPO in hepatopancreas

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Discussion

A new full-length 3040 bp cDNA sequence with 686 amino acids of ProPO were cloned from F. chinensis. This sequence has a high identity to ProPO gene from other two penaeid shrimp, P. monodon and P. semisulcatus. Similarly F. chinensis ProPO has a high sequence homology to other invertebrate ProPOs. All of which contain at least one conserved putative cell adhesion integrin-binding motif [20] The ProPO of both insects and branchiopods are copper-binding molecules that play important role in sclerotization of cuticle and encapsulation of foreign particles [21]. The domains found in F. chinensis ProPO were Hemocyanin-N, Hemocyanin-M and Hemocynin-C, which suggested that crustacean and insect prophenoloxidases were potential members of hemocyanin gene family [21, 22]. The entire hemocyanin gene family—hemocyanin, cryptocyanin, prophenoloxidase and hexamerins—may participate at various degrees in these two vital functions of molting animals (oxygen binding and molting) [22]. We found that even if the conservative domain of ProPO and hemocyanin shared high identities, their amino acid sequences showed a big difference, such as Hemocyanin (GenBank accession number CAB85965.1, CAA51880.1) and ProPO of L. vannamei (GenBank accession number AAW51360.1, ABX76968.1, ABL10871.1). Therefore, even if the primers and probe designed using the sequence from the hemocyanin domain, ProPO primers and probe were still specific for ProPO and would not detect the hemocyanin cRNA in real time PCR.

Phylogenetic analysis suggested that F. chinensis ProPO together with other shrimp prophenoloxidases (P. semisulcatus, P. monodon, L. vannamei and M. japonicus) were clustered into the same subgroup. ProPOs from lobsters (H. americanu and H. gammarus) and crayfish (P. clarkia and P. leniusculus) were placed into another subgroup. Branch 2 of ProPOs from two crabs (S. serrata and C. magister).

In this experiment, the mRNA expression analysis showed that the expression of ProPO in haemocytes was the highest among these three experimental tissues, which was consistent with previous reports [4, 9, 10, 23]. The distributions of ProPO were studied in three shrimp species [23–26] and two crab species [27, 28]. P. monodon was reported that ProPO mRNA is synthesized in the hemocytes and not in the hepatopancreas [23], so did the giant freshwater prawn [10]. In L. vannamei, ProPO expression were widely detected in haemocytes, gill, heart, lymphoid organ, stomach, midgut, anterior midgut caecum and ganglion. A lower expression level was found in hepatopancreas, muscle and cuticular epidermis. And ProPO transcripts, however, were also detected in non-haemocyte cells, including F and E cells of the hepatopancreas, epithelium of stomach and anterior midgut caecum [24–26]. The ProPO of mud crab Scylla serrata was strongly expressed in haemocytes, but not in heart, eyestalk, gill, muscle, ovary, hepatopancreas, stomach, and intestine [27]. But in Chinese mitten crab the mRNA transcripts of EsProPO and PO specific activities were detected in all the examined tissues with the highest level in hepatopancreas [28].

The mRNA levels of ProPO were detected as lower in haemocytes after V. anguillarum injection 6 hpi, then increased at 12 hpi and reached the highest level at 72 hpi. In the lymphoid organ, the ProPO level was up-regulated to fourfold of normal at 6 hpi and dropped slowly to normal level at 72 hpi. All the control groups in three tissues had slighter change than that in the infection groups at all infection time, which implied that V. anguillarum injection stimulated a remarkable increase of ProPO mRNA level in haemocytes and in lymphoid organ. Our observation is also to an earlier report in white shrimp L. vannamei [29] The ProPO level in the V. anguillarum-injected animals appeared that crustaceans rely primarily on their innate immune response to protect themselves from a variety of pathogens. Therefore, the data suggest that the animals try to clear the infected invader at early hours after injection and there is an up regulation of ProPO expression. As time progresses, the host defense and ProPO expression can be significantly down-regulated due to infection.

Further studies are currently underway to investigate ProPO protein expression in vivo, under stimulation by V. anguillarum and by chemically modified siRNA, to determine whether the proteins expression level is consistent with the mRNA level.