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Abstract: As a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but has not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E. sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was 2247-bp with a 1539-bp open reading frame (ORF) encoding a 512-amino acid protein. The protein contained a 116-residue death domain (DD) at its N-terminus and a 272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4 proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in the ovary and testis, and moderately expressed in the thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2 (November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (β-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis. [Copyright &y& Elsevier]

Abstract: As pattern recognition receptors (PRRs), C-type lectins (CTLs) play significant roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (EsLecD) was identified from the crustacean Eriocheir sinensis. The cloning of full-length EsLecD cDNA was based on the initial expressed sequence tags (ESTs) isolated from a hepatopancreatic cDNA library. The full-length EsLecD cDNA of 686 bp with an open reading frame of 468 bp encodes a putative protein of 155 aa residues, including an N-terminal signal peptide and a single carbohydrate-recognition domain (CRD). By quantitative RT-PCR analysis, the EsLecD transcript was mainly detected in the hepatopancreas but rarely in other tissues, and it was significantly upregulated in the hepatopancreas after immune challenge with lipopolysaccharides. The recombinant EsLecD protein (rEsLecD) exhibited the ability to bind to all tested microorganisms, including bacteria and yeast. Meanwhile, calcium significantly increased the binding affinity of rEsLecD toward microorganisms, but it was not essential. The binding of rEsLecD induced the aggregation of microbial pathogens. Moreover, rEsLecD was capable of inhibiting the growth of microorganisms and even directly killing bacteria. Interestingly, rEsLecD could stimulate cellular encapsulation in vitro. In conclusion, results of this study suggest that EsLecD acts as an antibacterial PRR participating in the innate immunity of invertebrates. [Copyright &y& Elsevier]

Pattern recognition receptors (PPRs) are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD). EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS) injection. The recombinant EsLecF (rEsLecF) protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate. [ABSTRACT FROM AUTHOR]

Pattern recognition receptors (PPRs) are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD). EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS) injection. The recombinant EsLecF (rEsLecF) protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: The basic mechanism of host fighting against pathogens is pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. For this reason, we investigated the immune functionality of two pattern recognition receptors, C-type lectin EsLecA and EsLecG, post lipopolysaccharides (LPS) challenge in Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The cloning of full-length EsLecA and EsLecG cDNA were based on the initial expressed sequence tags (EST) isolated from a hepatopancreatic cDNA library via PCR. The EsLecA cDNA contained a 480-bp open reading frame that encoded a putative 159-amino-acid protein, while EsLecG cDNA contained a 465-bp open reading frame that encoded a putative 154-amino-acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of carbohydrate recognition domains that were common among C-type lectin superfamilies. EsLecA and EsLecG mRNA expression in E. sinensis were (a) both detected in all tissues, including the hepatopancreas, gills, hemocytes, testis, accessory gland, ovary, muscle, stomach, intestine, heart, thoracic ganglia and brain, and (b) responsive in hepatopancreas, gill, hemocytes post-LPS immuno-challenge all appeared dramatically variation. Collectively, the data presented here demonstrate the successful isolation of two novel C-type lectins from the Chinese mitten crab, and their role in the innate immune system of an invertebrate. [Copyright &y& Elsevier]

C-type lectins are one family of pattern recognition receptors (PRRs) that play important roles in innate immunity. In this work, cDNA and genomic sequences for a new C-type lectin ( FcLec5) were obtained from the Chinese white shrimp Fenneropenaeus chinensis. FcLec5 cDNA contains an open reading frame of 1,008 bp and its genomic sequence is 1,137 bp with 4 exons and 3 introns. The predicted FcLec5 protein contains a signal peptide and two carbohydrate recognition domains (CRDs). The N-terminal CRD of FcLec5 has a predicted carbohydrate recognition motif of Gln-Pro-Asp (QPD), while the C-terminal CRD contains a motif of Glu-Pro-Gln (EPQ). Northern blot analysis showed that FcLec5 mRNA was specifically expressed in hepatopancreas. FcLec5 protein was expressed in hepatopancreas and secreted into hemolymph. Real-time PCR showed that FcLec5 transcript exhibited different expression profiles after immune-challenged with Vibrio anguillarum or White Spot Syndrome Virus (WSSV). Recombinant FcLec5 and its two individual CRDs could agglutinate most bacteria tested, and the agglutinating activity was Ca-dependent. Besides, the agglutinating activity to gram-negative bacteria is higher than that to gram-positive bacteria. Direct binding assay showed that recombinant FcLec5 could bind to all microorganisms tested (five gram-positive and four gram-negative bacteria, as well as yeast) in a Ca-independent manner. Recombinant FcLec5 also directly bound to bacterial peptidoglycan, lipopolysaccharide and lipoteichoic acids. These results suggest that FcLec5 may act as a PRR for bacteria via binding to bacterial cell wall polysaccharides in Chinese white shrimp. [ABSTRACT FROM AUTHOR]

Background: Insects react against pathogens through innate immunity. The cotton bollworm Helicoverpa armigera (H. armigera) is an important defoliator and an extremely destructive pest insect of many crops. The elucidation of the mechanism of the immune response of H. armigera to various pathogens can provide a theoretical basis for new approaches to biologically control this pest. Results: Four kinds of pathogens Bacillus thuringiensis, Klebsiella pneumoniae, Candida albicans, and Autographa californica multiple nucleocapsid nucleopolyhedrovirus harbored green fluorescence protein and polyhedron (AcMNPV-GFP) were used to challenge the insect. The cellular and humoral immune responses to the pathogens were analyzed in the challenged H. armigera. The results show that in the five kinds of haemocytes, only granulocytes phagocytized the Gram-negative and Gram-positive bacteria and fungi. All haemocytes can be infected by AcMNPV. Fourteen immune-related genes including pattern recognition receptors (PRRs) such as peptidoglycan recognition proteins (HaPGRP and HaPGRP C) and Gram-Negative Bacteria-Binding Protein (HaGNBP), and antimicrobial peptides (AMPs) such as cecropin-1, 2 and 3 (HaCec-1, 2 and 3), lysozyme (HaLys), attacin (HaAtt), gallerimycin-like (HaGall), gloverin-like (HaGlo), moricin-like (HaMor), cobatoxin-like (HaCob), galiomicin-like (HaGali), and immune inducible protein (HaIip) appeared in different expression profiles to different pathogen infections. The transcripts of 13 immune related genes (except HaPGRPC) are obviously up-regulated by Gram-positive bacteria. HaCec-1 and 3, HaMor, HaAtt, HaLys, HaIip, HaPGRP and HaGNBP are greatly up-regulated after fungal infection. HaGNBP, HaCec-2, HaGall, HaGlo, HaMor, HaCob, HaGali obviously increased in Gram-negative bacterial infection. Only five genes, HaGNBP, HaCec-1, HaGali, HaGlo, and HaLys, are weakly up-regulated after viral infection. The AMP transcripts had higher expression levels than the PRR transcripts after the microbial challenge. Conclusions: These data suggest that the granulocytes are the major phagocytes in H. armigera. All haemocytes can be infected by AcMNPV. The transcripts of 14 immune related genes have different expression patterns in H. armigera infected by different pathogens, which means that the immune-related genes may have different functions against various kinds of pathogens. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Lysozymes possess antibacterial activities, making them crucial defense proteins in innate immunity. In this study, a chicken-type (c-type) lysozyme (designated PcLyzc) was cloned and characterized from red swamp crayfish Procambarus clarkii . The full-length cDNA had an open reading frame of 435 base pairs encoding a polypeptide of 144 amino acid residues. Multiple alignments and phylogenetic analysis revealed that PcLyzc shared high similarity to the other known invertebrate c-type lysozymes. PcLyzc transcripts were steadily expressed in a wide range of tissues in healthy crayfish, and were prominently up-regulated in the hepatopancreas and gills after Vibrio anguillarum or Aeromonas hydrophila challenge. Recombinant PcLyzc showed inhibitory activity in vitro against both Gram-positive bacteria, including Staphylococcus aureus, Micrococcus luteus and Bacillus thuringiensis , and Gram-negative bacteria, including A. hydrophila , V. anguillarum and Escherichia coli . By overexpressing PcLyzc through introducing exogenous recombinant protein, or silencing PcLyzc expression through injecting double strand RNA, it was found that PcLyzc could help eliminate the invading bacteria in crayfish hemolymph and could protect crayfish from death, possibly by promoting the hemocytic phagocytosis. These results indicated that PcLyzc played a role in the antibacterial immunity of crustaceans, and laid a foundation of developing new therapeutic agents in aquaculture. [ABSTRACT FROM AUTHOR]

Highlights: [•] Identifying a new antibacterial peptide astacidin from crayfish Procambarus clarkii. [•] PcAst could bind to bacterial cell wall components. [•] PcAst displayed inhibitory activity against bacterial growth. [•] PcAst functioned in the bacterial clearance immune reaction. [•] PcAst has an important function in antibacterial innate immune response. [Copyright &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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 ( Mj SRC). The mRNA level of Mj SRC 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 Mj SRC have the ability of binding different bacteria and glycans in vitro . After knockdown of Mj SRC, the bacterial clearance ability and phagocytic rate of hemocyte decreased significantly in vivo . Meanwhile, overexpression of Mj SRC in shrimp enhanced the clearance ability and phagocytic rate of hemocytes. Further study found that Mj SRC could regulate the expression of several antimicrobial peptides (AMPs). All these results indicate that Mj SRC plays important roles in antibacterial immunity in kuruma shrimp by enhancing hemocyte phagocytosis and AMP expression. [ABSTRACT FROM AUTHOR]

Down syndrome cell adhesion molecule (Dscam) mediates innate immunity against pathogens in arthropods. Here, a novel Dscam from red claw crayfish Cherax quadricarinatus (CqDscam) was isolated. The CqDscam protein contains one signal peptide, ten immunoglobulin domains, six fibronectin type III domains, one transmembrane domain and cytoplasmic tail. CqDscam phylogenetically clustered with other invertebrate Dscams. Variable regions of CqDscam in N-terminal halves of Ig2 and Ig3 domains, complete Ig7 domain and TM domain can be reshuffled after transcription to produce a deluge of >37,620 potential alternative splice forms. CqDscam was detected in all tissues tested and abundantly expressed in immune system and nerve system. Upon lipopolysaccharides (LPS) and b-1, 3-glucans (Glu) challenged, the expression of CqDscam was up-regulated, while no response in expression occurred after injection with peptidoglycans (PG). Membrane-bound and secreted types of CqDscam were separated on the protein level, and were both extensively induced post LPS challenge. Membrane-bound CqDscam protein was not detected in the serum, but localized to the hemocyte surface by immuno-localization assay. In the antimicrobial assays, the recombinant LPS-induced isoform of CqDscam protein displayed bacterial binding and growth inhibitory activities, especially with Escherichia coli . These results suggested that CqDscam, as one of pattern-recognition receptors (PRRs), involved in innate immune recognition and defense mechanisms in C. quadricarinatus , possibly through alternative splicing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: Ras-related protein Rap GTPase has been implicated in cell adhesion, cell proliferation, and cell junction formation. The first shrimp Rap cDNA (FcRap) was recently identified from the Chinese white shrimp Fenneropenaeus chinensis. The full length of FcRap is 1013bp, with a 561bp open reading frame that encodes a 186 amino acid protein. FcRap has a calculated molecular mass of 20.90kDa and pI of 6.37. Phylogenetic analysis shows that FcRap and other Rap proteins are clustered into one group. Results from the quantitative real-time polymerase chain reaction show that FcRap could be detected mainly in the hemocytes, hepatopancreas, stomach, and gills, whereas a relatively lower expression level could be detected in the heart and intestines. FcRap in the hemocytes was upregulated 2h post Vibrio challenge, and it was upregulated 2h post white spot syndrome virus (WSSV) challenge, and peaked at 6h before it declined at 12h. No variation in the FcRap transcript was observed in the gills under the Vibrio challenge, but it was initially downregulated 2h post WSSV challenge, and then it was upregulated and peaked at 6h before it was eventually went down at 12h. The rFcRap protein was successfully expressed in Escherichia coli BL21DE3. The pull-down analysis showed that rFcRap protein could interact with VP28, an envelope protein of WSSV. The probable roles of Rap GTPase in shrimp innate immunity are presented for the first time. [Copyright &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: 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 [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 12h. FcLegu mRNA in hepatopancreas was down-regulated by Vibrio. FcLegu transcript first declined from 2h to 6h and then recovered from 12h to 24h 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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 473bp and 574bp, 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 575bp 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 &y& Elsevier]

Abstract: Thioredoxin (TRX) is involved in cell redox homeostasis. In addition, it is responsible for maintaining proteins in their reduced state. In our study, a Fenneropenaeus chinensis thioredoxin (FcTRX) gene was identified from the Chinese white shrimp. The full length of FcTRX was 777 bp, including a 60 bp 5′ untranslated region (UTR), a 318 bp open reading frame (ORF) encoding a 105 amino acids protein, and a 399 bp 3′ UTR. FcTRX contained a TRX domain with a conserved motif of Cys-Gly-Pro-Cys (CGPC). No signal peptide was predicted by SMART analysis. The molecular mass and pI of FcTRX were 12 kDa and 4.62, respectively. FcTRX is a widely distributed gene, and its mRNA is detected in hemocytes, hearts, hepatopancreas, gills, stomach, and intestine from an unchallenged shrimp. The expression level of FcTRX was the highest in hepatopancreas, where it was down-regulated to the lowest level at 12 h white spot syndrome virus (WSSV) challenge. In the gills, it went up to the highest level at 6 h. Western blot showed that FcTRX protein in hepatopancreas challenged with WSSV was down-regulated from 2 h to 12 h and then restored to the level similar to that of unchallenged shrimp at 24 h. In the gills challenged with WSSV, the FcTRX protein was up-regulated from 6 h to 24 h. Our research indicated its possible role in the anti-WSSV innate immunity of shrimps. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this study, we report a Kazal-type serine proteinase inhibitor, named hcPcSPI1, from freshwater crayfish (Procambarus clarkii). We found that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099) or became decreased after a white spot syndrome virus (WSSV) challenge. In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not α-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPI1 was also found to possess bacteriostatic activity against the B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The whey major component, whey acidic protein (WAP), has one or more WAP domains characterized by a four-disulfide core (4-DSC) structure. These kinds of proteins are involved in multiple functions, including proteinase inhibitor activity, antimicrobial activity, ATPase inhibitor activity, and regulatory function in cell proliferation. Recent research indicates that WAP domain-containing proteins play an important role in the innate immunity of crustaceans. In this study, a novel double WAP domain (DWD)-containing protein named Fc-DWD was found for the first time in Chinese white shrimp, Fenneropenaeus chinensis. The open reading frame of Fc-DWD encodes a protein of 117 amino acids, including a signal peptide of 16 amino acids and two WAP domains. The predicted molecular mass of the mature protein is 12.78kDa with an estimated pI of 8.49. The first WAP domain, named WAP 1, composed of 49 amino acids locates in the amino-terminal of Fc-DWD, and the second WAP domain, named WAP 2, composed of 45 amino acids locates in the carboxy-terminal. Fc-DWD mRNA was upregulated in hemocytes, hepatopancreas, gills, and stomach of bacteria- and virus-challenged shrimp. Results of the binding assay showed that rFc-DWD could bind to both Gram-negative bacteria and Gram-positive bacteria. rWAP 1 could only bind to Gram-positive bacteria, but rWAP 2 could bind to both Gram-negative and positive bacteria. Moreover, rFc-DWD exhibited proteinase inhibitory activity against the secretory proteinase(s) from Bacillu s subtilis and Pseudomonas aeruginosa. All of these findings suggest that Fc-DWD may play an important role in enabling the host defense to execute its proteinase inhibitory activity against pathogens. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: C-type lectins play important roles in innate immunity of invertebrates. In the present study, we report a novel C-type lectin, named FcLec4, from the Chinese white shrimp Fenneropenaeus chinensis. FcLec4 contains a single carbohydrate recognition domain (CRD) with a putative signal peptide. Phylogenetic analysis indicated that FcLec4 was distant from most reported C-type lectins from shrimps. The expression of FcLec4 increased at both mRNA and protein level after stimulation of Vibrio anguillarum. Recombinant FcLec4 could agglutinate both Gram-positive and -negative bacteria in the presence of calcium. The recombinant protein could bind to peptidoglycan and selectively bind to microorganisms. Interestingly, the tight binding of recombinant FcLec4 to V. anguillarum might facilitate the subsequent clearance of the bacteria in vivo. To the best of our knowledge, this might be the first report that a C-type lectin was found to be directly involved in the anti-V. anguillarum response in shrimps. [Copyright &y& Elsevier]

Abstract: Four trypsin-like serine proteases, designated as Fctry1, Fctry2, Fctry3, and Fctry4, were obtained from the hepatopancreas of the Chinese shrimp, Fenneropenaeus chinensis. Three trypsin-like serine proteases, specifically Fctry1, 2, and 3 were observed to have the conserved catalytic triad (H, D, and S). Regarding Fctry4, the catalytic triad S was substituted by F, and the mature peptide was found to be inactive. Further analysis for similarities indicated that Fctry1, 2 and 4 were 89–92% identical to trypsins from Pacific white shrimp (Litopenaeus vannamei); while Fctry3 was only 42% identical to trypsins from salmon louse (Lepeophtheirus salmonis). The genomic organizations of Fctry1, 2 and 4 are also quite different from Fctry3. So, Fctry3 may be a new member of the trypsin-like serine protease family. Moreover, a semi-quantitative reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR was carried out to analyze the distribution profiles and expression patterns after they were subjected to a bacterial and viral challenge. The results showed that the four trypsin-like serine proteases were upregulated in the hepatopancreas of shrimp infected with the white spot syndrome virus (WSSV), and Fctry3 increased after a bacteria challenge. Therefore, these trypsin-like serine proteases might be involved in the innate defense reactions against different pathogens in Chinese shrimp. [Copyright &y& Elsevier]

Abstract: With their inherent immune system, shrimps survive in marine environments despite the presence of different stressors, such as bacteria, fungi, and virus. In order to fight against and subsist even in the presence of such pathogens, the shrimps'' ability to produce and release antimicrobial peptides are indeed important. In view of this, a new type of genetic material, an antimicrobial peptide containing a single whey acidic protein domain (SWD), was cloned from the haemocytes of fleshy prawns (Fenneropenaeus chinensis). Hence, this peptide is named Fc-SWD. The full-length cDNA of Fc-SWD was 399 bp, and it contained a single open reading frame (ORF) of 273 bp encoding an Fc-SWD of 90 amino acids, including a putative 24-amino acid signal peptide and a mature 66 residue peptide. Sequence comparison and phylogenetic analysis of SWDs and other whey acidic protein (WAP) domains containing proteins in crustaceans suggested that the crustacean WAP containing proteins can be divided into four classes: crustin I, crustin II, SWD, and carcinin and carcinin-like proteins. Apparently, the Fc-SWD is a new type in the SWD subgroup. Northern blot analysis demonstrated that Fc-SWD transcripts were constitutively expressed in the haemocytes of non-infected shrimps and increased in the haemocytes of infected shrimps. Meanwhile, Fc-SWD mRNA was not detected in the hearts, hepatopancreas, stomachs, gills, intestines, or ovaries of the non-infected shrimps nor the infected ones. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that the Fc-SWD signal was detected in the haemocytes, hearts, and gills of both the unchallenged and the challenged shrimps, and in the stomach of the challenged shrimps. Western blot analysis indicated that the Fc-SWD protein appeared to be synthesized in the haemocytes with a signal peptide and then secreted to the haemolymph as a mature peptide when the F. chinensis was infected by pathogens. Thus, the Fc-SWD (for the full-length peptide including the signal peptide), the mFc-SWD (for the mature peptide), and the Fc-WAPD (for the single WAP domain) were manifested in the Escherichia coli, respectively. Further findings indicated that the purified recombinant Fc-SWD and the mFc-SWD have antimicrobial activities against Gram-positive (G+) and Gram-negative (G−) bacteria and fungi, as well as a strong inhibitory activity against subtilisin A and protein K. The Fc-WAPD has anti-protease activities and low antimicrobial activity. In view of the foregoing findings, it can be concluded that Fc-SWD is one of the factors that affect shrimps'' immunity against various pathogens. [Copyright &y& Elsevier]

Abstract: Lectins play important roles in animal innate immune responses by serving as pattern recognition receptors, opsonins, or effector molecules. Here, we report a novel hepatopancreas-specific C-type lectin, designated Fc-hsL, from the hepatopancreas of the Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-hsL is 571bp long with a 480bp open reading frame that encodes a 159-residue protein. Fc-hsL contains a signal peptide and a single C-type lectin-like domain (CTLD) or carbohydrate recognition domain (CRD). It has an EPN(Glu-Pro-Asn) motif with a predicted ligand-binding site specific for mannose. Fc-hsL was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated following challenge of shrimp with bacteria or virus. Fc-hsL was not detected in other tissues but was induced in the stomach of immune-challenged shrimp. Fc-hsL protein was detected in both hemolymph and the hepatopancreas of bacteria- and virus-challenged shrimp. Recombinant mature Fc-hsL has no hemagglutinating activity, but calcium-dependent agglutinating activity against some Gram-positive and Gram-negative bacteria was detected. The rFc-hsL also has binding activity to some Gram-positive and Gram-negative bacteria and high antimicrobial activity against some bacteria and fungi. These in vitro functions of recombinant Fc-hsL were calcium-independent. Fc-hsL may act as a pattern recognition receptor in antibacterial defense and as an effector in innate immunity of Chinese shrimp. [Copyright &y& Elsevier]