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The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) typically composing of eight subunits (CSN1-8) mediates the process of deneddylation and deubiquitination. The fifth subunit of COP9 signalosome, CSN5, has special characteristics compared with the other seven subunits, and plays vital roles in the deneddylation activity and diverse cellular processes. However, the role of CSN5 in antiviral immunity is not clear. In this study, we identified 8 subunits (CSN1-8) of COP9 signalosome in shrimp Marsupenaeus japonicus. CSN1-6 were existed in all tested tissues, but CSN7-CSN8 were not detected in hepatopancreas. After WSSV challenged, the expression level of Csn1 to Csn4, and Csn6 to Csn8 were highly decreased, but the expression level of Csn5 was conspicuously increased in shrimp challenged by white spot syndrome virus (WSSV). The CSN5 was recombinantly expressed in Escherichia coli and its polyclonal antibody was prepared. The expression level of CSN5 was conspicuously increased at RNA and protein levels in the shrimp challenged by WSSV. After knockdown of Csn5 by RNA interference, the WSSV replication was obviously increased in shrimp. When injected the recombinant protein of CSN5 with the membrane penetrating peptide into shrimp, WSSV replication was inhibited and the survival rate of shrimp was significantly improved compared with control. We further analyzed the expression of antimicrobial peptides (AMPs) in Csn5-RNAi shrimp, and the results showed that the expression of several AMPs was declined significantly. These results indicate that CSN5 inhibits replication of WSSV via regulating expression of AMPs in shrimp, and the recombinant CSN5 might be used in shrimp aquaculture for the white spot syndrome disease control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F 1 F 0 -ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Whey acidic protein domain (WAPD) is a usual motif in crustaceans, and is found mainly in the immune-related proteins. In the present study, a protein containing three tandem WAPDs was identified in red swamp crayfish Procambarus clarkii and designated as PcTWD. This is the first report of a protein of such domain architecture in crustaceans. Introducing the WAPDs of PcTWD into phylogenetic analysis led to the classification of crustacean WAP proteins into classical crustins and proteins containing solely WAPDs. PcTWD was widely expressed in multiple tissues, including hemocytes, gills, hepatopancreas, heart, stomach and intestine. Its expression could be significantly induced by Staphylococcus aureus or Aeromonas hydrophila challenge. Knockdown PcTWD expression by RNAi suppressed host resistance against A. hydrophila, while exogenous recombinant PcTWD could enhance the host immunity. The three WAPDs showed a labor division. The first two domains were responsible for the protease inhibitory activity, and the third domain contributed to the antimicrobial activity. Thus PcTWD was found as an important protein in crayfish antibacterial immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Thymosins β are actin-binding proteins that play a variety of different functions in inflammatory responses, wound healing, cell migration, angiogenesis, and stem cell recruitment and differentiation. In crayfish, thymosins participate in antiviral immunology. However, the roles of thymosin during bacterial infection in shrimp remain unclear. In the present study, four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus, and named as Mjthymosin2, Mjthymosin3, Mjthymosin4, and Mjthymosin5 according the number of their thymosin beta actin-binding motifs. Mjthymosin3 was selected for further study because its expression level was the highest in hemocytes. Expression analysis showed that Mjthymosin3 was upregulated in hemocytes after challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant Mjthymosin3 protein could inhibit the growth of certain bacteria in an in vitro antibacterial test. Mjthymosins could facilitate external bacterial clearance in shrimp, and were beneficial to shrimp survival post V. anguillarum or S. aureus infection. The results suggested that Mjthymosins played important roles in the antibacterial immune response of kuruma shrimp., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Myeloid leukemia factor (MLF) plays an important role in development, cell cycle, myeloid differentiation, and regulates the RUNX transcription factors. However, the function of MLF in immunity is still unclear. In this study, an MLF was identified and characterized in kuruma shrimp Marsupenaeus japonicus, and named as MjMLF. The full-length cDNA of MjMLF contained 1111 nucleotides, which had an opening reading frame of 816 bp encoding a protein of 272 amino acids with an MLF1-interacting protein domain. MjMLF could be ubiquitously detected in different tissues of shrimp at the transcriptional level. The expression pattern analysis showed that MjMLF could be upregulated in shrimp hemocytes and hepatopancreas after white spot syndrome virus challenge. The RNA interference and protein injection assay showed that MjMLF could inhibit WSSV replication in vivo. Flow cytometry assay showed that MjMLF could induce hemocytes apoptosis which functioned in the shrimp antiviral reaction. All the results suggested that MjMLF played an important role in the antiviral immune reaction of kuruma shrimp. The research indicated that MjMLF might function as a novel regulator to inhibit WSSV replication in shrimp., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Scavenger receptors (SRs) comprise a large family of structurally diverse glycoproteins located on the cell membrane and function as pattern-recognition receptors (PRRs) participating in innate immunity in different species. Class C scavenger receptor (SRC) has been only identified in invertebrates and its biological functions still need to be researched. In this study, we characterized the anti-bacterial function of a SRC from kuruma shrimp Marsupenaeus japonicus (MjSRC). The mRNA level of MjSRC was up-regulated significantly in hemocytes of kuruma shrimp challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant extracellular domains (MAM and CCP domains) of MjSRC have the ability of binding different bacteria and glycans in vitro. After knockdown of MjSRC, the bacterial clearance ability and phagocytic rate of hemocyte decreased significantly in vivo. Meanwhile, overexpression of MjSRC in shrimp enhanced the clearance ability and phagocytic rate of hemocytes. Further study found that MjSRC could regulate the expression of several antimicrobial peptides (AMPs). All these results indicate that MjSRC plays important roles in antibacterial immunity in kuruma shrimp by enhancing hemocyte phagocytosis and AMP expression., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Leucine rich repeat (LRR) motif exists in many immune receptors of animals and plants. Most LRR containing (LRRC) proteins are involved in protein-ligand and protein-protein interaction, but the exact functions of most LRRC proteins were not well-studied. In this study, an LRRC protein was identified from kuruma shrimp Marsupenaeus japonicus, and named as MjLRRC1. MjLRRC1 was consistently expressed in different tissues of normal shrimp with higher expression in gills and stomach. At the transcriptional level, there were no significant changes of MjLRRC1 after injection of Vibrio anguillarum or Staphylococcus aureus in gills and hepatopancreas. While in V. anguillarum oral infection, MjLRRC1 was upregulated in stomach but not in intestine. The recombinant MjLRRC1 protein could bind to Gram-positive and Gram-negative bacteria, bacterial cell wall components including peptidoglycan, lipoteichoic acid, and lipopolysaccharide. MjLRRC1 regulated the expression of some antimicrobial peptide (AMP) genes and participated in bacteria clearance of stomach. All these results suggested that MjLRRC1 might play important roles in antibacterial immune response of kuruma shrimp., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The suppressor of cytokine signaling (SOCS) family is a kind of negative regulators in the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in mammals and Drosophila. In kuruma shrimp, Marsupenaeus japonicus, SOCS2 is identified and its expression can be stimulated by peptidoglycan and polycytidylic acid. However, if SOCS2 participates in regulating Jak/Stat pathway in shrimp still needs further study. In this study, SOCS2 with Src homology 2 domain and SOCS box was identified in kuruma shrimp, M. japonicus. SOCS2 existed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine, the expression of SOCS2 was upregulated significantly in the hemocytes and intestine of shrimp challenged with Vibrio anguillarum at 6 h. To analyze SOCS2 function in shrimp immunity, bacterial clearance and survival rate were analyzed after knockdown of SOCS2 in shrimp challenged with V. anguillarum. Results showed that bacterial clearance increased, and the survival rate improved significantly comparing with controls. The SOCS2 was expressed in Escherichia coli and the recombinant SOCS2 was injected into shrimp, and Stat phosphorylation and translocation were analyzed. The result showed that "overexpression" of SOCS2 declined Stat phosphorylation level and inhibited Stat translocation into the nucleus. After knockdown of SOCS2 in shrimp prior to V. anguillarum infection, the expression level of antimicrobial peptides, including anti-lipopolysaccharide factors C1, C2 and D1, and Crustin I was upregulated significantly, and the expression of the AMPs was declined after recombinant SOCS2 injection. The SOCS2 expression was also decreased in Stat-knockdown shrimp challenged by V. anguillarum at 6 and 12 h. Therefore, SOCS2 negatively regulates the AMP expression by inhibiting Stat phosphorylation and translocation into nucleus in shrimp, meanwhile, SOCS2 expression was also regulated by Jak/Stat pathway., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Lysin motif (LysM) is a peptidoglycan and chitin-binding motif with multiple functions in bacteria, plants, and animals. In this study, a novel LysM and putative peptidoglycan-binding domain-containing protein was cloned from kuruma shrimp (Marsupenaeus japonicus) and named as MjLPBP. The cDNA of MjLPBP contained 1010 nucleotides with an open reading frame of 834 nucleotides encoding a protein of 277 amino acid residues. The deduced protein contained a Lysin motif and a transmembrane region, with a calculated molecular mass of 31.54 kDa and isoelectric point of 8.61. MjLPBP was ubiquitously distributed in different tissues of shrimp at the mRNA level. Time course expression assay showed that MjLPBP was upregulated in hemocytes of shrimp challenged with Vibrio anguillarum or Staphylococcus aureus. MjLPBP was also upregulated in hepatopancreas after white spot syndrome virus and bacteria challenge. The recombinant protein of MjLPBP could bind to some Gram-positive and Gram-negative bacteria and yeast. Further study found that rMjLPBP bound to bacterial cell wall components, including peptidoglycans, lipoteichoic acid, lipopolysaccharide, and chitin. The induction of several antimicrobial peptide genes and phagocytosis-related gene, such as anti-lipopolysaccharide factors and myosin, was depressed after knockdown of MjLPBP. MjLPBP could facilitate V. anguillarum clearance in vivo. All the results indicated that MjLPBP might play an important role in the innate immunity of shrimp., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Intestinal innate immune response is an important defense mechanism of animals and humans against external pathogens. The mechanism of microbiota homeostasis in host intestines has been well studied in mammals and Drosophila. The reactive oxygen species (ROS) and antimicrobial peptides have been reported to play important roles in homeostasis. However, how to maintain the microbiota homeostasis in crustacean intestine needs to be elucidated. In this study, we identified a novel catalase (MjCAT) involved in ROS elimination in kuruma shrimp, Marsupenaeus japonicus. MjCAT mRNA was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine. After the shrimp were challenged with pathogenic bacteria via oral infection, the expression level of MjCAT was upregulated, and the enzyme activity was increased in the intestine. ROS level was also increased in the intestine at early time after oral infection and recovered rapidly. When MjCAT was knocked down by RNA interference (RNAi), high ROS level maintained longer time, and the number of bacteria number was declined in the shrimp intestinal lumen than those in the control group, but the survival rate of the MjCAT-RNAi shrimp was declined. Further study demonstrated that the intestinal villi protruded from epithelial lining of the intestinal wall were damaged by the high ROS level in MjCAT-knockdown shrimp. These results suggested that MjCAT participated in the intestinal host-microbe homeostasis by regulating ROS level., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Crustins are a family of cationic, cysteine-rich antimicrobial peptides with a whey acidic protein (WAP) domain in the C-terminal. They have diverse functions in antimicrobial immune responses. Four groups of crustins (crustins I, II, III, and IV) have been identified in crustaceans, but type I crustins have not been reported in penaeid shrimp until now. In this study, we identified four crustins in kuruma shrimp Marsupenaeus japonicus, and named them MjCrus I-2, 3, 4 and 5. These four crustins belong to type I crustins, which contain a signal peptide, cysteine-rich region at the N-terminus, and WAP domain at the C-terminus. Tissue distribution demonstrated that MjCrus I-2, 3 and 5 had high expression levels in hemocytes, gills and stomach. whereas MjCrus I-4 was distributed in all tissues detected. MjCrus I-2 to 5 showed different expression patterns in different tissues after Gram-positive bacterial (Staphylococcus aureus), Gram-negative bacterial (Vibrio anguillarum), and white spot syndrome virus (WSSV) challenge. The expression of MjCrus I-2 to 5 was upregulated by bacterial or WSSV challenge. The three crustins were recombinantly expressed in Escherichia coli, and the purified proteins showed few antimicrobial activities. Three MjCrus Is could bind to different bacteria. MjCrus I-2 and 3 showed different inhibitory abilities to secreted bacterial proteases. MjCrus I-4 could not inhibit bacterial proteases. After knockdown of MjCrus I-3, the bacterial scavenging ability to V. anguillarum was impaired. These results suggested that type I crustins played an important role in the innate immunity of shrimp., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fibrinogen-related proteins (FREPs) in invertebrates have important functions in innate immunity. In this study, the cDNA of FREP was identified from the kuruma shrimp Marsupenaeus japonicus (MjFREP2). The full-length cDNA of MjFREP2 is 1138 bp with an open reading frame of 954 bp that encodes a 317-amino acid protein comprising a signal peptide and a fibrinogen-like domain. MjFREP2 could be detected in hemocytes, heart, hepatopancreas, gills, stomach, and intestines. MjFREP2 could also be upregulated in hemocytes after Vibrio anguillarum and Staphylococcus aureus challenge. Agglutination and binding assay results revealed that the recombinant MjFREP2 bound to bacteria and polysaccharides. Immunocytochemical analysis results showed that MjFREP2 proteins were mainly distributed in the cytoplasm of hemocytes from unchallenged shrimp and transported to the membrane or secreted out of the cell after V. anguillarum or S. aureus challenge. The secreted MjFREP2 bound to the bacteria presented in shrimp hemolymph. The overexpression of MjFREP2 could enhance bacterial clearance by inducing the phagocytosis of hemocytes. This ability was impaired by knockdown of MjFREP2 with RNA interference. The cumulative mortality of MjFREP2-silenced shrimp was significantly higher than that of the control shrimp. These results suggested that MjFREP2 has an important function in the antibacterial immunity of M. japonicus., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Accumulating evidence suggests that peroxiredoxins (Prx) are key molecules in the pathogenesis of various infectious diseases and are potential therapeutic targets for major diseases such as cancers. In this study, we report a peroxiredoxin IV (Prx IV) in Marsupenaeus japonicus, designated as MjPrx IV, which exhibited peroxidase activity and participated in the anti-white spot syndrome virus (WSSV) immune response. MjPrx IV is a 245-amino acid polypeptide with a predicted 19-amino acid signal peptide, an Ahpc-TSA domain, and a 1-Cys PrxC domain. Phylogenetic analysis revealed that the protein belongs to the Prx IV subfamily. MjPrx IV transcripts were detected in the gills, hepatopancreas, heart, stomach, ovaries, spermary, and intestine tissues, and are upregulated in the gonads, gills and hemocytes of shrimp after WSSV challenge. The mature MjPrx IV peptide was recombinantly expressed in an Escherichia coli system. The protein exhibited peroxidase activity. Furthermore, dsRNA suppression of MjPrx IV increased WSSV replication in shrimp, whereas rMjPrx IV injection into shrimp decreased WSSV replication. These data suggest that MjPrx IV has an important role in shrimp antiviral immunity. To our knowledge, this study is the first to report a shrimp Prx IV that has anti-WSSV activity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

C-type lectins function as pattern recognition receptors and play important roles in the innate immune system of crustaceans. In this study, we reported a new CTL gene (designated as PcLec5) from red swamp crayfish, Procambarus clarkii. PcLec5 was mainly distributed in hepatopancreas, gills and intestine, and the PcLec5 transcripts were up-regulated in all the three tissues after challenge with bacteria Vibrio anguillarum. For further functional analyses, PcLec5 was recombinantly expressed in Escherichia coli and anti-PcLec5 polyclonal antiserum was prepared. The results of bacteria binding assay revealed that PcLec5 could selectively bind to 5 of 9 kinds of bacteria we used and had a tendency to bind to Gram-negative bacteria. Sugar binding assay showed that PcLec5 could bind to peptidoglycan, lipoteichoic acid and lipopolysaccharide, with the highest affinity to LPS. Furthermore, bacteria-clearance experiment showed PcLec5 could selectively facilitate the clearance of injected bacteria in crayfish, and the bacteria-clearance facilitating spectrum of PcLec5 was totally in agreement with its bacteria binding spectrum. These results suggested PcLec5 function as a pattern recognition receptor in crayfish immune system and had certain selectivity on bacteria pathogens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The single whey acidic protein (WAP) domain containing proteins (SWDs) in crustacean belong to type III crustins and have antiprotease activities and/or antimicrobial activities. Their functions in vivo in crustacean immunity need to be clarify. In this study, a new single WAP domain containing protein (SWD) was obtained from Marsupenaeus japonicus, designated as MjSWD. The full-length cDNA of MjSWD was 522 bp.The open reading frame of MjSWD encoded a protein of 79 amino acids, with a 24 amino acid signal peptide and a WAP domain. Tissue distribution analysis revealed that MjSWD transcripts were generally expressed in all the tested tissues, including hemocytes, heart, hepatopancreas, gill, stomach and intestine. The time course expression of MjSWD was analyzed by quantitative real time PCR, and the results exhibited that MjSWD was upregulated after bacteria (Vibrio anguillarum, Staphylococcus aureus) and white spot syndrome virus (WSSV) challenge in gills and stomach of the shrimp. The purified recombinant protein of MjSWD could bind to several Gram-negative and Gram-positive bacteria though binding to microbial polysaccharides (peptidoglycan). MjSWD could inhibit the activity of Subtilisin A and Proteinase K and bacteria-secreted proteases. The results of natural infection with MjSWD incubated bacteria showed that the inhibition of MjSWD against bacterial secreted proteases was contributed to inhibiting bacteria invasion and dissemination in the shrimp. The MjSWD is, thus, involved in the shrimp antibacterial innate immunity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

BAX inhibitor-1 (BI-1) was originally described as an anti-apoptotic protein in both animal and plant cells. BI-1 overexpression suppresses ER stress-induced apoptosis in animal cells. Inhibition of BI-1 activity could induce the cell death in mammals and plants. However, the function of BI-1 in crustacean immunity was unclear. In this paper, the full-length cDNA of a BI-1 protein in red swamp crayfish, Procambarus clarkii (PcBI-1) was cloned and its expression profiles in normal and infected crayfish were analyzed. The results showed that PcBI-1 was expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestines of the crayfish and was upregulated after challenged with Vibrio anguillarum and with white spot syndrome virus (WSSV). To determine the function of PcBI-1 in the innate immunity of the crayfish, the RNA interference against PcBI-1 was performed and the results indicated the hemocyte programmed cell death rate was increased significantly and WSSV replication was declined after PcBI-1 knocked down. Altogether, PcBI-1 plays an anti-apoptotic role, wherein high PcBI-1 expression suppresses programmed cell death, which is beneficial for WSSW replication in crayfish., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

C-type lectins play important roles in the innate immune system of crustaceans. In this study, a novel C-type lectin gene, designated as PcLec4, was obtained from the red swamp crayfish (Procambarus clarkii). Quantitative real-time polymerase chain reaction revealed that PcLec4 is mainly expressed in the crayfish hepatopancreas and intestine, and the PcLec4 mRNA expression is upregulated after challenged with the bacteria Vibrio anguillarum. PcLec4 was recombinantly expressed in Escherichia coli and anti-PcLec4 polyclonal antiserum was prepared. Binding experiments revealed that the recombinant PcLec4 binds to various bacteria and polysaccharides on the bacterial surface, which suggests that PcLec4 recognizes bacterial pathogens. Overexpression of PcLec4 in crayfish using the pIeLec4 vector was performed. The results show that the crayfish overexpressing PcLec4 eliminate injected V. anguillarum more quickly than the control, which suggests that PcLec4 elicits further immune response for removing invading bacteria. The results of the survival experiment confirmed the function of PcLec4 in resisting V. anguillarum because PcLec4 overexpression in crayfish significantly increased the crayfish survival rate. These results reveal that PcLec4 has an important role in the antibacterial immunity of crayfish, and in vivo PcLec4 overexpression might be used as a disease control strategy in aquiculture., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Invertebrates, including shrimp, have developed very complicated innate immune system against pathogens. Much work has been performed on the innate immunity of shrimp, including immune recognition, signal transduction, effector molecules and antiviral responses due to its great economic value. Pattern recognition is the first step of innate immunity. Pattern recognition receptors (PRRs) sense the presence of infection and activate immune responses. The studies on shrimp PRRs revealed the recognition mechanism of shrimp at a certain degree. To date, 11 types of pattern recognition receptors (PRRs) have been identified in shrimp, namely, β-1,3-glucanase-related proteins, β-1,3-glucan-binding proteins, C-type lectins, scavenger receptors, galectins, fibrinogen-related proteins, thioester-containing protein, Down syndrome cell adhesion molecule, serine protease homologs, trans-activation response RNA-binding protein and Toll like receptors. A number of PRRs have been functionally studied and have been found to have different binding specificities and immune functions. The present review aims to summarize the current knowledge on the PRRs of shrimp., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cathepsin C (Cath C) is a lysosomal cysteine protease that belongs to the papain superfamily. Cath C is capable of activating many chymotrypsin-like serine proteases and is reported to be a central coordinator for the activation of many serine proteinases in immune and inflammatory cells. In this study, Cath C cDNA was cloned from Fenneropenaeus chinensis (Fc). The complete cDNA of Fc-Cath C in Chinese white shrimp was found to be 1445-base pairs (bp) long. It contained an open reading frame (ORF) 1356-bp long and encoded a 451-amino acid residue protein, including a 17-amino acid residue signal peptide. Real-time PCR analysis results indicated that Fc-Cath C was present in all the tissues detected and exhibited high level of transcription in the hepatopancreas. In hemocytes, hepatopancreas, gills and intestine, Fc-Cath C was upregulated after stimulation by the Vibrio anguillarum and the white spot syndrome viruses (WSSVs). Replication of the WSSV increased after the injection of Fc-Cath C antiserum or knockdown Cath C by RNA interference. These results implied that Cath C might play a crucial role in the antiviral immune response of shrimp., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Fibrinogen-related proteins play important roles in innate immunity. We isolated a fibrinogen-related protein gene (MjFREP1) in kuruma shrimp Marsupenaeus japonicus. MjFREP1 encoded a protein of 270 amino acids, including a 223 amino acid fibrinogen-like domain. Quantitative real-time polymerase chain reaction analysis shows that MjFREP1 is mainly expressed in the gills and the expression is significantly upregulated by Vibrio anguillarum, Staphylococcus aureus, or white spot syndrome virus (WSSV) challenge. Recombinant MjFREP1 fibrinogen-like domain agglutinates Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis, Bacillus megaterium, and S. aureus in the presence of calcium ions. The fibrinogen-like domain of MjFREP1 binds peptidoglycans, LPS, bacteria, and the VP28 of WSSV. These results suggest that the MjFREP1 may play an important role in the shrimp immune response against different pathogens., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Thioesterase superfamily member 2 (Them2) is a single hotdog fold thioesterase domain-containing protein. Its biological function is not well known. Recently, a hotdog fold thioesterase (FcThem) was cloned for the first time from the Chinese white shrimp. The full length of FcThem is 748bp. It encodes a protein with 142 amino acids with a predicted molecular mass of 14.79kDa and an isoelectric point of 8.76. No signal peptide was predicted. Multiple alignment of FcThem with other Them2 proteins suggested a conserved HGG motif. Phylogenetic analysis showed that FcThem were clustered with vertebrate Them2 protein into one group. The RT-PCR results showed that FcThem was a widely distributed gene and could be detected in the hemocytes, heart, hepatopancreas, gills, stomach, intestines, and ovaries of unchallenged shrimps. In hemocytes, its transcript was upregulated 24h post WSSV challenge. In the gills, the FcThem went up at a 6h WSSV challenge. FcThem expression in the ovaries was also affected by the WSSV and was increased after the 2h WSSV challenge, reaching the highest level at 6h. Our results show that FcThem probably has roles in the innate immunity system of shrimps and investigations will be carried out to explore this finding further., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Serine proteases (SPs) and serine protease homologs (SPHs) participate in digestion, embryonic development, blood coagulation, and immune defense responses. In this paper, we identify one SP and two SPHs, including a masquerade SPH (FcMas), a CUB domain containing SP (FcCUBSP), and a single domain containing SPH (FcSPH2) in Chinese white shrimp, Fenneropenaeus chinensis. FcMas has a Gly-rich region formed by three repeats of LGGQGGG, a clip domain and a C-terminal SP-like domain. Absence of Ser catalytic residue results in the loss of serine protease activity of FcMas, which then functions as an SPH. FcCUBSP has a signal peptide, followed by a CUB domain and an SP domain. FcSPH2 has a signal peptide and an SP-like domain. Loss of one catalytic residue (H) makes FcSPH2 catalytically inactive, which is considered an SPH. Phylogenetic analysis shows that FcMas and other SPHs from shrimp or insect are classified into one group. FcSPH2 is grouped in the chymotrypsin family. RT-PCR results show that FcMas mRNA is mainly distributed in hemocytes and gills. FcCUBSP is only detected in gills, whereas FcSPH2 is found in hepatopancreas only. QRT-PCR is used to analyze changes of FcMas, FcCUBSP and FcSPH2 in some tissues challenged with white spot syndrome virus (WSSV) or Vibrio. FcMas in hemocytes is down-regulated by WSSV or Vibrio challenge, and down-regulated by WSSV in gills. However, it is up-regulated upon Vibrio challenge in gills. FcCUBSP in gills and FcSPH2 in hepatopancreas are up-regulated upon WSSV or Vibrio challenge. Results suggest the roles of FcMas, FcCUBSP and FcSPH2 in shrimp's innate immunity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

C-type lectins are abundantly found in crustaceans. They function in the immune system by recognizing, opsonizing, or agglutinating. Some of them also feature anti-microbial activity. In this study, we identify a hepatopancreas-specific C-type lectin (PcLec2) that responds significantly to immune challenges in red swamp crayfish (Procambarus clarkii). Recombinant PcLec2 shows no agglutinating or anti-microbial activity. It can bind to lipopolysaccharides and bacterial cells in the absence of calcium, and its binding to Gram-negative bacteria is stronger than that to Gram-positive ones. Moreover, the protein can stimulate the activation of prophenoloxidase both in vitro and in vivo. We conclude that this C-type lectin may be an upstream detector of the prophenoloxidase activating system in crayfish., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The anti-lipopolysaccharide factors (ALFs) are a group of effector molecules of innate immunity in arthropods, exhibiting binding and neutralizing activities to lipopolysaccharides. In this study, an ALF cDNA sequence (PcALF1) was identified from red swamp crayfish, Procambarus clarkii. The deduced peptide of PcALF1 was conserved; it manifested the signal peptide and lipopolysaccharide (LPS)-binding domain, especially the two conserved cysteine residues at both ends of the domain. Transcripts of PcALF1 were detected in multiple tissues. Results of quantitative real-time PCR exhibited that the expression level of PcALF1 was induced by virus and Gram-positive and Gram-negative bacteria. Purified recombinant protein of PcALF1 revealed multiple biological activities: it gave all the tested bacteria and fungi a tight binding; it could bind microbial polysaccharides (LPS, LTA, and β-glucan) as well. In vitro, the antimicrobial activity assay was demonstrated as a broad spectrum against Gram-positive and Gram-negative bacteria and a fungus. The rPcALF1 also exhibited a clearance activity on Vibrio anguillarum in a dose-dependent manner in vivo., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Lysozyme is an important immune effector and is widely distributed in many organisms. In the present study, two novel invertebrate-type lysozymes (Pclysi1 and Pclysi2) were cloned from red swamp crayfish, Procambarus clarkii. Alignment analysis showed that these two genes were different in catalytic residues. Results of RT-PCR showed that these two genes share similar tissue distribution patterns, and both were upregulated after bacteria challenge. The mature recombinant proteins were expressed in Escherichia coli system and purified. Activity analysis revealed that rPclysi1 had no muramidase activity and isopeptidase activity, but had antimicrobial activity. Meanwhile, rPclysi2 was muramidase-deficient and had no antimicrobial activity, but possessed isopeptidase activity. Above data suggest that Pclysi1 and 2 may offer different functions in the crayfish immunity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The cDNAs encoding CathL and legumain from Chinese white shrimp Fenneropenaeus chinensis (FcCathL, FcLegu) were obtained. Both FcCathL and FcLegu mRNA were expressed mainly in the hepatopancreas of unchallenged shrimp. Time-course analysis of FcCathL showed that FcCathL was upregulated in the hepatopancreas of shrimp challenged with white spot syndrome virus (WSSV) at 12 h. FcLegu mRNA in hepatopancreas was down-regulated by Vibrio. FcLegu transcript first declined from 2 h to 6 h and then recovered from 12 h to 24 h in hepatopancreas challenged with WSSV. FcCathL protein was detected in the hemocytes, hepatopancreas, gill, stomach, and intestine of unchallenged shrimp. Three bands of FcCathL protein detected in some tissues may represent preproenzyme, single chain and mature double chain form respectively. In hepatopancreas, FcLegu was detected in the proenzyme form. In other tissues, only active form could be detected. The protein of FcLegu was down-regulated by Vibrio or WSSV challenge in the stomach and gills. FcCathL and FcLegu were proposed to play a role in shrimp innate immunity for the first time., (Copyright 2010 Elsevier Ltd. All rights reserved.)

White spot syndrome virus (WSSV) infects all shrimp species and is the greatest detriment to shrimp culture. To better understand the mechanism of molecular responses to WSSV infection in Chinese white shrimp Fenneropenaeus chinensis, two dimensional electrophoresis (2-DE) was used. Differentially expressed proteins in the hepatopancreas of control and WSSV-injected Chinese white shrimp between (6, 12 and 24 h post-injection) were screened. Quantitative intensity analysis and mass spectrometry revealed that 54 spots of 47 proteins were significantly up-regulated, including some immune-related proteins, such as Toll receptor precursor, Leu-rich repeat protein, peroxinectin and serine proteinase-like protein. Fourteen spots of 13 proteins, such as heat shock protein, ATP synthase sub-unit beta and thrombospondin, were down-regulated in WSSV-infected shrimps. Protein expression patterns of the infected shrimp were drastically altered by WSSV infection. Some of the altered proteins were further investigated at the mRNA level using semi-quantitative reverse transcript PCR. These data may provide some information about shrimp proteins that participate in the WSSV infection process., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Three Kazal-type serine proteinase inhibitors, hcPcSPI2, hpPcSPI3, and hpPcSPI4, with complete cDNA sequences, were identified from a cDNA library of the red swamp crayfish, Procambarus clarkii. Semi-quantitative RT-PCR shows that hcPcSPI2 exists mainly in hemocytes while both hpPcSPI3 and hpPcSPI4 were detected in the hepatopancreas and the heart. Homology comparison and phylogenic analysis indicate that hpPcSPI3 and hpPcSPI4 shared high identity and formed the same group, and both of them were different from other hepatopancreas type inhibitors in crustaceans forming a large group, while hcPcSPI2 as well as other hemocyte type inhibitors belonged to another cluster. In addition, the temporal expression profiles of these three inhibitors were studied with quantitative real-time PCR and the results suggest that hcPcSPI2 and hpPcSPI3 are likely to be involved in antiviral immune response, and all these three inhibitors respond to Vibrio anguillarum challenge in different degrees. Further study was done on hcPcSPI2. Western blot demonstrates that hcPcSPI2 only exists in semigranular cells. Besides, after V. anguillarum challenge, the hcPcSPI2 protein could also be detected in cell-free hemolymph. Subsequently, the biochemical characteristics and bacteriostatic activity of hcPcSPI2 were assayed. The results indicate that hcPcSPI2 shows weak inhibitory activity against subtilisin A and trypsin, and may trigger bacteriostatic activity towards Bacillus subtilis and Bacillus thuringiensis, possessing MIC(50) of 30.4 and 25.0 microM, respectively. These studies reveal that hcPcSPI2 may also play an important role in the anti-bacterial immunity of the crayfish., (2010 Elsevier Ltd. All rights reserved.)

Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences (Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473 bp and 574 bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575 bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Single WAP domain-containing proteins (SWDs) are small proteins possessing a whey acidic protein (WAP) domain at the C-terminal region. In this study, a complementary deoxyribonucleic acid (cDNA) of SWD-containing protein was isolated from the hemocytes of red swamp crayfish, Procambarus clarkii called Pc-SWD. The full-length cDNA sequence is 998 bp. The deduced amino acid sequence consists of 74 residues with a signal peptide of 20 residues. The mature peptide has a single WAP domain which contains eight conserved cysteine residues forming a "four-disulphide core" (4-DSC). The predicted molecular mass of the mature protein is 5.97 kDa, with an estimated pI of 7.71. Tissue distribution analysis by reverse-transcribed polymerase chain reaction (RT-PCR) revealed that Pc-SWD transcripts were primarily found in the hemocytes, heart, hepatopancreas, gills, and intestine. The results of time course analysis demonstrated that expression of Pc-SWD was decreased at 6 h followed by a significant upregulation from 48 h to 72 h in hemocytes after white spot syndrome virus (WSSV) injection. A similar expression pattern was found in the hepatopancreas after WSSV injection. In addition, Pc-SWD expression was visibly upregulated in the gills from 6 h to 72 h after WSSV injection. The results of Western bolt revealed that Pc-SWD was constitutively expressed in the heart, hepatopancreas, gills, and intestine of unchallenged crayfish. A weak band was detected in the hemocytes and hemolymph of unchallenged P. clarkii. The Pc-SWD expression was upregulated in the hemocytes and gills after challenging with WSSV; however, no obvious change occurred in the heart and intestine. rPc-SWD could bind to both Gram-negative and -positive bacteria strongly. Moreover, rPc-SWD exhibited specific proteinase inhibitory activity against the secretory proteinase(s) from B. subtilis and P. aeruginosa. All these findings suggest that Pc-SWD possibly functions as an immunity effector in defense against the invasion of crayfish pathogens., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Acyl-CoA-binding protein (ACBP) and fatty acid-binding protein (FABP) are involved in lipid metabolism. ACBP plays a key role in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, vesicular trafficking, and gene regulation. In our study, a 536 bp cDNA of ACBP (FcACBP) was cloned and identified as a widely distributed gene in the Chinese white shrimp, Fenneropenaeus chinensis. Its expression in intestine was upregulated in response to white spot syndrome virus (WSSV) or Vibrio anguillarum infection. The expression patterns were confirmed by Western blot analysis. FABPs, members of the lipid-binding protein superfamily, play an important role in lipid metabolism and also participate in vertebrate innate immunity. A cDNA of FABP (FcFABP) cloned from the hepatopancreas of the shrimp was 715 bp in size and encoded a 14 kDa protein. FcFABP appeared to be a basic fatty acid binding protein with a predicted isoelectric point of 9.16. It showed sequence similarity to both vertebrate and invertebrate FABPs. Phylogenetic analysis showed that FcFABP, together with LvFABP, were clustered into one group. FcFABP was detected mainly in the hepatopancreas and expression level increased after a challenge with WSSV. FcFABP was down-regulated by V. anguillarum challenge. The protein also had bacterial binding activity. These two lipid metabolism related proteins may play important roles in shrimp innate immunity.

In invertebrates, the Toll signaling pathway is important for activation of antimicrobial peptides in the innate immune system. Activation of the Toll pathway requires binding of Toll with its ligand Spätzle. Here we described a Spätzle-like protein, designated as Fc-Spz, from hemocytes of Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequence of Fc-Spz shares 54% identity with Spätzle-like protein of salmon louse (Lepeophtheirus salmonis). Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), quantitative real time PCR, and Western blot analyses were carried out to analyze the expression pattern and distribution profile of Fc-Spz in shrimp after challenged with bacteria and virus. The results showed that Fc-Spz mRNA was up-regulated in all the tissues tested in shrimp injected with Vibrio anguillarum and white spot syndrome virus (WSSV). The C-terminal active Fc-Spz domain (114 residues) was expressed in Escherichia coli and purified by affinity chromatography. The recombinant Fc-Spz C-114 was injected into crayfish (Procambarus clarkii) to determine the expression levels of several antimicrobial peptide genes. The results showed that recombinant Fc-Spz C-114 could up-regulate crustin 2 expression in crayfish. These results suggest that Fc-Spz may play a role in the innate immune defence of Chinese shrimp and crayfish.

C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this work, a cDNA for a new C-type lectin, FcLec3, was obtained from Chinese white shrimp Fenneropenaeus chinensis using expressed sequence tag analysis and rapid amplification of the cDNA ends. FcLec3 contains an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that FcLec3 was mainly expressed in hepatopancreas and that the expression of FcLec3 was obviously up-regulated by Vibrio anguillarum or white spot syndrome virus (WSSV) challenge. Recombinant FcLec3 could agglutinate Gram-negative and -positive bacteria with the presence of calcium. A following agglutination inhibitory test indicated that FcLec3 could recognize muramic acid and peptidoglycan. Besides, pull-down assay showed that the recombinant protein could interact with VP28, one major envelope protein of WSSV. These results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp.

Serine proteinase inhibitor plays an essential role in arthropods by restraining the activities of endogenic or exogenic serine proteinases. Four Kazal-type serine proteinase inhibitors, Fcspi-1-4, from the hepatopancreas of Chinese white shrimp, Fenneropenaeus chinensis, were cloned and identified. The open reading frames (ORFs) of Fcspis are 1389, 1236, 1080, and 939 base pairs, encode the pre-proteins of 462, 411, 359, and 312 amino acids and form the 9, 8, 7, and 6 typical Kazal domains, respectively. When analyzing the amino acid sequences of the four inhibitors, it was found that they might have been derived from the same transcript, which was subjected to alternative splicing, and none of the Kazal domains were identical within each inhibitor. Multiple alignments showed that the Kazal inhibitors were homologous with a conserved motif of Cx(3)Cx(6)VCGSDGxTYx(3)CxLx(5)Cx(5)ITx(6)GC. The results from RT-PCR indicated that the expression of Fcspis as a whole was upregulated by bacterial challenge, no obvious change was noticed after viral challenge, and Fcspi-1 had a similar expression pattern with that of Fcspis. Recombinant FcSPIs were successfully expressed in bacteria and purified for further study. Recombinant FcSPI-1 was sensitive to DTT and had thermal stability. The inhibitory kinetics assay suggested that rFcSPI-1 was a mixed-type fast tight binding inhibitor with inhibitory activities against subtilisin A at a molar ratio of 1:1, 1:2 against proteinase K, and 2:1 against elastase. It can firmly bound to two Gram-positive and one Gram-negative bacteria but without anti-bacterial ability. In addition, it inhibited the activities of both bacterial-secreted proteinases and natural chymotrypsin of Chinese white shrimp, suggesting that FcSPI-1 may participate in the immune defence response by inhibition of bacterial pathogen proteinases and possibly be involved in the regulation of shrimp proteinase activity.

Clip domain serine proteases and their homologs are involved in invertebrate innate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, cell adhesion, and melanization. Recognition of pathogens by pattern recognition receptors can trigger activation of a serine protease cascade. We report here the cDNA cloning of a serine protease (FcSP) and a serine protease homolog (FcSPH) from Chinese white shrimp, Fenneropenaeus chinensis. Both FcSP and FcSPH possess a clip domain at the N-terminal and an SP or SP-like domain at the C-terminal. In contrast to FcSP, FcSPH lacks a catalytic residue and is catalytically inactive. Tissue distribution and time course qRT-PCR analysis indicates that FcSP and FcSPH can respond to Vibrio anguillarum challenge in hemocytes, hepatopancreas and intestine. In situ hybridization analysis shows that FcSP is distributed in hemocytes and gills, and originated mainly from the hemocytes. FcSPH protein is expressed in gills and stomach of non-challenged shrimp. Its expression in gill mainly originates from the hemocytes in it. Two immunoreactive bands of FcSP can be detected in gills and stomach of non-challenged shrimp. FcSP protein is partially cleaved in non-challenged shrimp, while FcSPH protein is unprocessed in unchallenged shrimp and is partially cleaved after V. anguillarum challenge. Our results suggest that this Clip domain serine protease and its homolog may be involved in the serine protease cascade and play an important role in innate immunity of the shrimp.

A new member of the serine protease (SP) chymotrypsin (designated Fc-chy) was isolated from the hepatopancreas of Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA of Fc-chy contained 951 nucleotides with a polyadenylation sequence and a polyA tail. It encoded a peptide of 271 amino acids with a signal peptide of 17 amino acids and an activation peptide of 28 amino acids. The mature peptide concludes 226 amino acids. It contained the conserved catalytic triad (H, D, and S). Similarity analysis showed that Fc-chy shared high identity with chymotrypsins from the Pacific white shrimp, Litopenaeus vannamei. Northern blot, quantitative real-time PCR, in situ hybridization, and western blot analysis were carried out to analyze the expression pattern and distribution profiles of Fc-chy after bacteria and virus challenges. The results showed that Fc-chy transcription and Fc-chy protein levels were upregulated in the hepatopancreas after bacterial and viral infection. Fc-chy from the hepatopancreas was purified by affinity chromatography. It showed high hydrolytic activity toward the substrate N-succinyl-ala-ala-pro-phe p-nitroanilide (AAPF), and its activity was inhibited by Kazal-type SP inhibitor from Chinese shrimp. All of these may indicate that Fc-chy is involved in the innate immune reactions in Chinese shrimp.

O-methyltransferase (OMT) is ubiquitously present in diverse organisms and plays an important regulatory role in plant and animal growth, development, reproduction and defence and has also been implicated in human emotion and disease. A putative o-methyltransferase (OMT) gene has been cloned from the haemocytes of bacteria-infected Chinese shrimp (Fenneropenaeus chinensis) by suppression subtractive hybridisation (SSH) coupled with the SMART cDNA method. The isolated 944 bp full-length cDNA contains a single 666bp open reading frame (ORF) encoding a putative OMT protein of 221 amino acids. The predicted protein has a molecular weight of 24,572.06 Da and a pI of 5.27 as well as ten phosphorylation sites. Northern blot and in situ hybridisation analyses demonstrated that the OMT transcripts were constitutively expressed in tissue of shrimp challenged by bacterial infection and in unchallenged shrimp tissue. Constitutive OMT transcript was found in areas such as haemocytes, heart, hepatopancreas, stomach, gill, intestine and ovary. However, the OMT transcripts were upregulated in hepatopancreas and stomach in challenged shrimp.

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a approximately 6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use.

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal sequence (1-19) followed by a mature peptide (20-71). The sequence identity with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The mature peptide, with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp, and that Ch-penaeidin was constitutively expressed mainly in haemocytes.