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Invertebrates rely on innate immunity, including humoral and cellular immunity, to resist pathogenic infection. Previous studies showed that forkhead box transcription factor O (FOXO) participates in mucosal immune responses of mammals and the gut humoral immune regulation of invertebrates. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from shrimp (Marsupenaeus japonicus) and found that it was expressed at relatively basal levels in normal shrimp, but was upregulated significantly in shrimp challenged by Vibrio anguillarum. FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of the immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity. In the nucleus, activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. Taken together, our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by activating the IMD pathway in normal shrimp, and directly and indirectly promoting AMP expression and enhancing phagocytosis of hemocytes against pathogens in bacteria-infected shrimp. This study revealed the different functions of FOXO in the mucosal (local) and systemic antibacterial immunity of invertebrates. Author summary: Shrimp aquaculture is one of the world's fastest growing industries for producing animal proteins and has made a significant contribution to meeting the worldwide increased demand for animal proteins. However, disease outbreaks in aquaculture result in large economic losses to the industry. Studies of shrimp immune mechanisms could provide new strategies for disease prevention and control. The forkhead box transcription factor O family proteins (FOXOs) are involved in various critical biological process of organisms. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from kuruma shrimp (Marsupenaeus japonicus) and found that it played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity and directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. This study revealed the different functions of FOXO in the innate antibacterial immunity of invertebrates. [ABSTRACT FROM AUTHOR]

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein level. Knockdown of MjpIgR, and bloking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are characterized by the presence of a C-type carbohydrate recognition domain (CTLD) that by recognizing microbial glycans, is responsible for their roles as pattern recognition receptors in the immune response to bacterial infection. In addition to the CTLD, however, some CTLs display additional domains that can carry out effector functions, such as the collagenous domain of the mannose-binding lectin. While in vertebrates, the mechanisms involved in these effector functions have been characterized in considerable detail, in invertebrates they remain poorly understood. In this study, we identified in the kuruma shrimp (Marsupenaeus japonicus) a structurally novel CTL (MjCC-CL) that in addition to the canonical CTLD, contains a coiled-coil domain (CCD) responsible for the effector functions that are key to the shrimp’s antibacterial response mediated by antimicrobial peptides (AMPs). By the use of in vitro and in vivo experimental approaches we elucidated the mechanism by which the recognition of bacterial glycans by the CTLD of MjCC-CL leads to activation of the JAK/STAT pathway via interaction of the CCD with the surface receptor Domeless, and upregulation of AMP expression. Thus, our study of the shrimp MjCC-CL revealed a striking functional difference with vertebrates, in which the JAK/STAT pathway is indirectly activated by cell death and stress signals through cytokines or growth factors. Instead, by cross-linking microbial pathogens with the cell surface receptor Domeless, a lectin directly activates the JAK/STAT pathway, which plays a central role in the shrimp antibacterial immune responses by upregulating expression of selected AMPs. [ABSTRACT FROM AUTHOR]

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjβ-arrestin2. Further studies found that Mjβ-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus. [ABSTRACT FROM AUTHOR]

Highlights: [•] EsDorsal was cloned and characterized. [•] EsDorsal was highly expressed in immune tissues. [•] EsDorsal expression in crab hemocytes varied after pathogenic stimulation. [•] EsDorsal may be involved in immune responses against bacteria and fungi. [Copyright &y& Elsevier]

Abstract: Be absent of adaptive immunity which have both specificity and memory, invertebrates seem to have evolved alternative adaptive immune strategies to resist various intruding pathogens. Whereas vertebrates could generate a wide range of immunological receptors with somatic rearrangement, invertebrates possibly depend on alternative splicing of pattern-recognition receptors (PRRs). Recently, it has been suggested that a member of the immunoglobulin superfamily (IgSF), Down syndrome cell adhesion molecule (Dscam), plays a crucial role in the alternative adaptive immune system of invertebrates. At present, we successfully isolated and characterized the first crab Dscam from Eriocheir sinensis. EsDscam has typical domain architecture compared with other Dscam orthologs, including one signal-peptide, 10 immunoglobulin (Ig) domains, 6 fibronectin type III domains (FNIII), one transmembrane domain (TM) and one cytoplasmic tail. We had detected four hypervariable regions of EsDscam in the N-terminal halves of Ig2 (25) and Ig3 domain (30), the complete Ig7 (18) and also the transmembrane domain (2), potentially generate 27,000 unique isoforms at least. Transcription of EsDscam were both a) detected in all tissues, especially in immune system, digestive system and nerve system; b) significantly induced in hemocytes post lipopolysaccharides (LPS), peptidoglycans (PG) and β-1, 3-glucans (Glu) injection. Importantly, we had detected membrane-bound and secreted Dscam isoforms in E. sinensis, and showed that secreted isoforms were extensively transcribed post different PAMPs challenge respectively. Results from immuno-localization assay revealed that EsDscam evenly distributed in the cell surface of hemocytes. These findings indicated that EsDscam is a hypervariable PRR in the innate immune system of the E. sinensis. [Copyright &y& Elsevier]

A 967-bp cDNA, encoding a 31 kDa lipase with an isoelectric point of 8.8, was cloned from Helicoverpa armigera. The gene was named Ha-lipase. The Ha-lipase transcript was present in the head-thorax, midgut and fat body but not in integument and haemocytes from 5th instar larvae, with higher expression levels in feeding larvae than that in molting larvae. In the midgut, transcription of the gene during the 5th instar larval period was lower than during the 6th instar period. While in the fat body, lipase was not found at the 6th instar stage but only at the 5th instar stage. The expression of Ha-lipase could be up-regulated by methoprene and down-regulated by methoxyfenozide (RH-2485) and 20-hydroxyecdysone (20E). [ABSTRACT FROM AUTHOR]

Nucleoside diphosphate kinase (NDPK) is a key metabolic enzyme that catalyzes the synthesis of non-adenine nucleoside triphosphate (NTP) by transferring the terminal phosphate between nucleoside diphosphate (NDP) and NTP. NDPK regulates a variety of eukaryotic cellular activities including cell proliferation, development, and differentiation. The ndpk cDNA was cloned from the hemocytes of the scallop Chlamys farreri and designated Cf-ndpk. The full-length sequence of Cf-ndpk consists of 715 bp encoding a polypeptide of 153 amino acids with a calculated molecular mass of 16927.52 daltons and p I of 7.64. The mRNA expression and distribution of Cf-ndpk in both bacterially challenged and unchallenged scallops were studied by Northern blotting and in situ hybridization. The results showed that Cf-ndpk transcripts were present in hemocytes, gill, adductor muscle, mantle, digestive gland, foot, and gonad, and the expression level increased in hemocytes after bacterial challenge. Recombinant Cf-NDPK expressed in Escherichia coli could transfer the terminal phosphate between UDP and ATP. The Cf-NDPK protein was present in all tested tissues including foot, adductor muscle, digestive gland, gonad, mantle, gill, and hemolymph. It was up-regulated in hemolymph after bacterial challenge. Taken together, these results suggest that NDPK may play roles in the innate immune response of scallop. [ABSTRACT FROM AUTHOR]

Helicoverpa hormone receptor 3 (HHR3) is a transcription factor involved in larval molting regulation. To investigate its function and the possibility to improve the insecticidal activity of AcMNPV, HHR3 was expressed in Bac-to-Bac system and an AcMNPV-GFP-HHR3-Polh+ virus was constructed after recovery of the polyhedrin gene. HHR3 expression in Sf21 cells was confirmed by immunofluorescence microscopy. A red color identifying HHR3 was observed using fluorescence microscopy; this confirmed that HHR3 was expressed in Sf21 cells by the introduction of the recombinant virus AcMNPV-GFP-HHR3-Polh+. The AcMNPV-GFP-HHR3-Polh+ infected the hemocytes of H. armigera larvae by hemocoele injection or per oral ingestion. Results showed that AcMNPV-GFP-HHR3-Polh+ resulted in incomplete ecdysis, slower growth and lower median survival time on the Helicoverpa larvae. This recombinant virus may aid in the development of an insecticide that can be used in the field. [ABSTRACT FROM AUTHOR]

Abstract: 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 ∼6100Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108mg/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. [Copyright &y& Elsevier]

Phagocytosis is a conserved cellular response among metazoans. Opsonins are some molecules that label targets to increase their susceptibility to phagocytosis. Opsonins are usually captured by receptors on the surface of phagocytes. Our previous study found the C-type lectin FcLec4 from Chinese white shrimp Fenneropenaeus chinensis might function as an opsonin to facilitate bacterial clearance. In the present study we purified the native FcLec4 protein and confirmed its opsonic activity in the near relation, kuruma shrimp Marsupenaeus japonicus. The possible receptor of FcLec4 was identified as β-integrin by panning a T7 phage display library of shrimp hemocytes and then confirmed by co-immunoprecipitation assay. We further proved that the interaction between FcLec4 and β -integrin did not rely on the carbohydrate recognition domain but on the N terminus of FcLec4. In addition, inhibition of FcLec4 expression using RNAi delayed bacterial clearance, and β -integrin knockdown suppressed the opsonic activity of FcLec4. This study is the first to show the direct interaction between an opsonin and its receptor in crustaceans. Our study provides new insights into invertebrate phagocytosis and the functions of C-type lectins. [ABSTRACT FROM AUTHOR]

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: Pattern recognition proteins (PRPs), such as lipopolysaccharide and β-1,3-glucan binding protein (LGBP), have been identified in many animals and play a crucial role in invertebrate defense systems. In the current study, an LGBP gene was cloned from fleshy prawn (Fenneropenaeus chinensis, Fc-LGBP) utilizing homology cloning and RACE methods. The full cDNA of the Fc-LGBP gene in fleshy prawn was 1253bp in size with a deduced 366 amino acid protein that includes a glycosyl hydrolase domain. Northern blot and RT-PCR data suggested that Fc-LGBP mRNA was mostly synthesized in haemocytes and that the expression was down-regulated 24h post-injection of bacteria. In situ hybridization demonstrated that Fc-LGBP mRNA was only detected in haemocyte cytoplasm, with no detection in other tissues. The molecular weight of the purified recombinantly expressed Fc-LGBP was approximately 46kDa. Immunohistochemistry of haemocytes revealed that Fc-LGBP protein was localized on the membrane of most cells. Data from bacterial binding assays utilizing purified protein suggested that rFc-LGBP had strong binding activity to Gram-negative bacteria. [Copyright &y& Elsevier]

β-Thymosins participate in numerous biological activities, including cell proliferation and differentiation, wound healing, and anti-inflammatory and antimicrobial activities. Many studies have investigated vertebrate β-thymosins, whereas few reports have focused on invertebrate β-thymosins. In this study, nine isoforms of β-thymosins ( Pc Thy-1 to Pc Thy-8) were identified from the red swamp crayfish Procambarus clarkii . The isoforms contained different numbers of the thymosin β actin-binding motif. Pc Thy-1 contained one thymosin β actin-binding motif, whereas Pc Thy-8 contained eight motifs. Western blot analysis with anti- Pc Thy-4 antibody showed that three to six isoforms were present in one tissue, and Pc Thy-4, Pc Thy-5, Pc Thy-6, and Pc Thy-7 were the main isoforms in several tissues. Time course expression analysis of Pc Thys at the protein level showed that Pc Thy-4 was upregulated in hemocytes and gills after white spot syndrome virus (WSSV) challenge. Pc Thy-4, which contained four thymosin β actin-binding motifs, was selected for further research. Tissue distribution analysis by quantitative real-time PCR showed that PcThy-4 was present in tissues of the hemocytes, heart, hepatopancreas, gills, stomach, and intestine at the transcriptional level. Transcriptional expression profiles showed that PcThy-4 was upregulated after WSSV challenge. In vivo RNAi and protein injection assay results showed that Pc Thy-4 inhibited the replication of WSSV in crayfish and enhanced the survival rate after WSSV infection. Furthermore, Pc Thy-4 promoted hemocyte phagocytosis of WSSV. Overall, results suggested that Pc Thys protected crayfish from WSSV infection and played an important role in antiviral immune response. [ABSTRACT FROM AUTHOR]

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 Mj Crus 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 Mj Crus I-2, 3 and 5 had high expression levels in hemocytes, gills and stomach. whereas Mj Crus I-4 was distributed in all tissues detected. Mj Crus 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 Mj Crus 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 Mj Crus Is could bind to different bacteria. Mj Crus I-2 and 3 showed different inhibitory abilities to secreted bacterial proteases. Mj Crus I-4 could not inhibit bacterial proteases. After knockdown of Mj Crus 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. [ABSTRACT FROM AUTHOR]

Crustins are cationic cysteine-rich antimicrobial peptides (AMPs) that contain multiple domains (glycine-rich, cysteine-rich, or proline-rich) at the N-terminus and whey acidic protein (WAP) domains at the C-terminus. Crustins have multiple functions, including protease inhibition and antimicrobial activity. Other functions of crustins need to be clarified. In this study, a novel crustin with a cysteine-rich region, and a single WAP domain, belonging to type I crustins, was identified in Marsupenaeus japonicus and designated as Mj Cru I-1. Mj Cru I-1 was expressed in various tissues. The expression of MjCru I-1 was upregulated in the hemocytes of shrimp challenged with bacteria. MjCru I-1 could bind to bacteria by binding to the cell wall molecules of the bacteria, such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipoteichoic acid (LTA). The synthesized WAP domain of Mj Cru I-1 but not synthesized Cys-rich domain has antibacterial and agglutinative activities. Scanning electron microscope assay showed that the bacterial cells treated with s Mj Cru I-1 appeared to be disrupted and cracked compared with those of the control samples. The knockdown of MjCru I-1 could reduce bacterial clearance and injection of Mj Cru I-1 could significantly increase the survival rate of shrimp infected with Vibrio anguillarum and Staphylococcus aureus compared with those of the control samples. Further study discovered that Mj Cru I-1 could increase the hemocyte phagocytosis against V. anguillarum and S. aureus . These results suggest that Mj Cru I-1 has dual functions, bactericidal and phagocytosis promoting activities, in the antibacterial immunity of shrimp. [ABSTRACT FROM AUTHOR]

Anti-lipopolysaccharide factors (ALFs) are a group of critical effector molecules with a broad spectrum of antimicrobial activities in crustaceans. Four groups of ALFs (A, B, C, and D) have been identified in peneaid shrimp. In the study, we identified a new group of ALFs (designated as Mj ALF-E) from Marsupenaeus japonicus . This new group (group E) included Mj ALF-E1 and E2. Mj ALF-E1 was highly expressed in hemocytes, heart, and intestine, whereas E2 was highly expressed in gills, stomach, and intestine. Expressions of both Mj ALF-E1 and E2 were upregulated by bacterial challenge. Synthesized LPS-binding domain peptides of Mj ALF-E1 and E2 strongly bind to bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN). The recombinant r Mj ALF-E2 showed relatively weak binding activity to LPS and PGN. Both synthesized peptides and r Mj ALF-E2 exhibited antimicrobial activity against Gram-negative bacteria, whereas r Mj ALF-E2 could promote the clearance of bacteria in vivo. After knockdown of Mj ALF-E2 and infection with Vibrio anguillarum , shrimp showed high and rapid mortality compared with GFPi shrimp. These results suggest that Mj ALF-Es serves a protective function against bacterial infection in shrimp. [ABSTRACT FROM AUTHOR]

Calnexin (Cnx) is an endoplasmic reticulum membrane–bound lectin chaperone that comprises a dedicated maturation system with another lectin chaperone calreticulin (Crt). This maturation system is known as the Cnx/Crt cycle. The main functions of Cnx are Ca 2+ storage, glycoprotein folding, and quality control of synthesis. Recent studies have shown that Cnx is important in phagocytosis and in optimizing dendritic cell immunity. However, the functions of Cnx in invertebrate innate immunity remain unclear. In this research, we characterized Cnx in the kuruma shrimp Marsupenaeus japonicus (designated as Mj Cnx) and detected its function in shrimp immunity. The expression of MjCnx was upregulated in several tissues challenged with Vibrio anguillarum . Recombinant Mj Cnx could bind to bacteria by binding polysaccharides. Mj Cnx protein existed in the cytoplasm and on the membrane of hemocytes and was upregulated by bacterial challenge. The recombinant Mj Cnx enhanced the clearance of V. anguillarum in vivo, and the clearance effects were impaired after silencing MjCnx with RNA interference assay. Recombinant Mj Cnx promoted phagocytosis efficiency of hemocytes. These results suggest that Mj Cnx functions as one of the pattern recognition receptors and has crucial functions in shrimp antibacterial immunity. [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]

Highlights: [•] MjLTL1 and MjADAM have similar distribution and expression patterns in M. japonicus. [•] Both MjLTL1 and MjADAM are upregulated by bacterial challenge. [•] MjLTL1 agglutinates and binds to different bacteria. [•] MjADAM might mediate the proteolytic release of extracellular domain of MjLTL1. [•] MjLTL1 enhanced the clearance of bacteria by promoting phagocytosis in vivo. [Copyright &y& Elsevier]

Abstract: Receptor for activated C kinase 1 (RACK1), which has seven tandem WD40 domains, is a scaffolding protein. RACK1 plays different roles by binding to different partner proteins. It is involved in hormone signaling and development, and now some evidence indicates it may have a role in innate immunity. In this paper, RACK1 cDNA from Chinese white shrimp (FcRACK1) was identified. The full length of the FcRACK1 gene is 1037bp, including a 30bp 5′UTR, a 957bp ORF encoding a 318 amino acid protein, and a 50bp 3′UTR with the polyadenylation sequence AATAAA and a poly (A) tail. The FcRACK1 protein is characterized by seven WD40 repeat domains; the ending two amino acids of each WD40 domain are WK, WD, WN, WS, WD, WD, and WQ, respectively. The length of each domain is between 30 and 44 amino acids. Multiple alignments of RACK1s showed that RACK1s are highly conserved. RT-PCR showed that FcRACK1 could be detected in hemocytes, the heart, hepatopancreas, gills, stomach, intestine, and ovary. FcRACK1 in hemocytes was down-regulated after a 2h WSSV challenge, and FcRACK1 in gills was up-regulated after a 2h Vibrio challenge. FcRACK1 in ovary went down after a 12h Vibrio challenge and then up-regulated at 24h. FcRACK1 in ovary was first down-regulated at 2h after a WSSV challenge and then up-regulated to the highest level at 6h. It finally went down from 12 to 24h. In hepatopancreas, FcRACK1 was also up-regulated by microbe challenge. Our results indicated its probable role in shrimp innate immunity. [Copyright &y& Elsevier]

Abstract: 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 MIC50 of 30.4 and 25.0 μM, respectively. These studies reveal that hcPcSPI2 may also play an important role in the anti-bacterial immunity of the crayfish. [Copyright &y& Elsevier]

Abstract: 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 &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: Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that modulate cell behavior in homeostasis and during development, wound-healing, immune response and tumor growth of adult tissues in vertebrates. In invertebrates these proteins are a major component of cortical rods in mature oocytes. A fragment of a thrombospondin-like gene was generated by screening a subtractive cDNA library constructed from the hemocytes of Chinese shrimp, Fennerpenaeus chinensis. The full length F. chinensis cDNA of thrombospondin was cloned by 3′- and 5′-rapid amplification of cDNA ends (3′- and 5′-RACE). The complete cDNA sequence, named Fc-TSP, is 2886bp and the open reading frame of the cDNA encodes a 938-residue protein that contains three ChtBD2 domains, an EGF domain, a TSP-3 domain and a common TSP-C (CTD) domain. The protein shares a high sequence identity with the mj-TSPa (46.3%), mj-TSPb (46.9%) and mj-TSPc (51.9%) of Marsupenaeus japonicus. The expression and distribution of Fc-TSP in both challenged and unchallenged shrimps were studied by Northern blot, RT-PCR and in situ hybridization. Northern blot analysis showed that the Fc-TSP transcripts were detected in the hemocytes, heart, intestine, stomach and ovary of both challenged and unchallenged shrimps, but the signal was much stronger in the challenged tissues. A strong hybridization signal was detected only in challenged hepatopancreas, with no signal in the unchallenged tissue. The RT-PCR showed that the Fc-TSP was detected in both challenged and unchallenged tissues including the hemocytes, heart, hepatopancreas, stomach, gills, intestine, spermary and ovary. Except for the ovary and spermary, the signal of challenged tissues was relatively stronger than that of unchallenged ones, especially in hepatopancreas. These results suggest that the thrombospondin was upregulated in the hemocytes, heart, intestine and stomach of challenged shrimp, and induced in the hepatopancreas of challenged shrimps. Therefore, Fc-TSP may be involved in the defense responses of the shrimp. [Copyright &y& Elsevier]