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AMPK plays significant roles in the modulation of metabolic reprogramming and viral infection. However, the detailed mechanism by which AMPK affects viral infection is unclear. The present study aims to determine how AMPK influences white spot syndrome virus (WSSV) infection in shrimp (Marsupenaeus japonicus). Here, we find that AMPK expression and phosphorylation are significantly upregulated in WSSV-infected shrimp. WSSV replication decreases remarkably after knockdown of Ampkα and the shrimp survival rate of AMPK-inhibitor injection shrimp increases significantly, suggesting that AMPK is beneficial for WSSV proliferation. Mechanistically, WSSV infection increases intracellular Ca2+ level, and activates CaMKK, which result in AMPK phosphorylation and partial nuclear translocation. AMPK directly activates mTORC2-AKT signaling pathway to phosphorylate key enzymes of glycolysis in the cytosol and promotes expression of Hif1α to mediate transcription of key glycolytic enzyme genes, both of which lead to increased glycolysis to provide energy for WSSV proliferation. Our findings reveal a novel mechanism by which WSSV exploits the host CaMKK-AMPK-mTORC2 pathway for its proliferation, and suggest that AMPK might be a target for WSSV control in shrimp aquaculture. AMPK and its downstream signaling pathways are implicated in the white spot syndrome virus (WSSV) response of the shrimp Marsupenaeus japonicus, with WSSV using this pathway to promote its proliferation. [ABSTRACT FROM AUTHOR]

Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture. Author summary: White spot syndrome virus (WSSV) is the causative pathogen of white spot disease (WSD) and represents the most destructive viral disease of shrimp. The virus has evolved various strategies to escape from host defenses or exploit host biological pathways for its reproduction. Studies on viral immune-escape mechanisms can provide new strategies for disease prevention and control in shrimp aquaculture. Mechanistic target of rapamycin (mTOR) plays a central role in the regulation of cell growth and metabolism, which nucleates two distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) with diverse functions at different levels of the signaling pathway. mTORC1 is reported to be exploited by viruses in their reproduction. However, the detail mechanism remains unclear. In this study, we identified a new mechanism of mTOR being hijacked by WSSV in shrimp (Marsupenaeus japonicus). WSSV infects shrimp by its receptor, pIgR and induces the interaction of the intracellular domain of pIgR with Calmodulin. Calmodulin subsequently promotes the activation of AKT by interaction with the pleckstrin homology domain of the kinase. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, S6Ks, for viral protein synthesis. Moreover, mTORC1 also phosphorylates 4EBP1, which results in the separation of 4EBP1 from eIF4E for the translation of viral proteins in shrimp. Our study reveals a novel strategy for WSSV proliferation in shrimp and indicates that the components of mTORC1 may represent potential clinical targets for WSSV control in shrimp aquaculture. [ABSTRACT FROM AUTHOR]

Lysyl oxidases (LOXs) are copper-dependent monoamine oxidases, and they play critical roles in extracellular matrix (ECM) remodeling. The LOX and LOX-like (LOXL) proteins also have a variety of biological functions, such as development and growth regulation, tumor suppression, and cellular senescence. However, the functions of LOXLs containing repeated scavenger receptor cysteine-rich (SRCR) domains in immunity are rarely reported. In this study, we characterized the antiviral and antibacterial functions of a lysyl oxidase-like (LOXL) protein containing tandem SRCR domains in Marsupenaeus japonicus. The mRNA level of LoxL was significantly upregulated in the hemocytes and intestines of shrimp challenged using white spot syndrome virus (WSSV) or bacteria. After the knockdown of LoxL via RNA interference, WSSV replication and bacterial loads were apparently increased, and the survival rate of the shrimp decreased significantly, suggesting that LOXL functions against pathogen infection in shrimp. Mechanistically, LOXL interacted with the envelope proteins of WSSV or with lipopolysaccharide and peptidoglycan from bacteria in shrimp challenged using WSSV or bacteria, and it promoted the expression of a battery of antimicrobial peptides (AMPs) via the induction of Dorsal nuclear translocation against viral and bacterial infection. Moreover, LOXL expression was also positively regulated by Dorsal in the shrimp challenged by pathogens. These results indicate that, by acting as a pattern recognition receptor, LOXL plays vital roles in antiviral and antibacterial innate immunity by enhancing the expression of AMPs in shrimp. [ABSTRACT FROM AUTHOR]

Trained immunity is driven by metabolism and epigenetics in innate immune cells in mammals. The phenomenon of trained immunity has been identified in invertebrates, including shrimp, but the underlying mechanisms remain unclear. To elucidate mechanisms of trained immunity in shrimp, the metabolomic changes in hemolymph of Marsupenaeus japonicus trained by the UV-inactivated white spot syndrome virus (UV-WSSV) were analyzed using tandem gas chromatography–mass/mass spectrometry. The metabolomic profiles of shrimp trained with UV-WSSV followed WSSV infection showed significant differences comparison with the control groups, PBS injection followed WSSV infection. 16 differential metabolites in total of 154 metabolites were identified, including D-fructose-6-phosphate, D-glucose-6-phosphate, and D-fructose-6-phosphate, and metabolic pathways, glycolysis, pentose phosphate pathway, and AMPK signaling pathway were enriched in the UV-WSSV trained groups. Further study found that histone monomethylation and trimethylation at H3K4 (H3K4me1 and H3K4me3) were involved in the trained immunity. Our data suggest that the UV-WSSV induced trained immunity leads to metabolism reprogramming in the shrimp and provide insights for WSSV control in shrimp aquaculture. [ABSTRACT FROM AUTHOR]

The gut microbiota is a complex group of microorganisms that is not only closely related to intestinal immunity but also affects the whole immune system of the body. Antimicrobial peptides and reactive oxygen species participate in the regulation of gut microbiota homeostasis in invertebrates. However, it is unclear whether nitric oxide, as a key mediator of immunity that plays important roles in antipathogen activity and immune regulation, participates in the regulation of gut microbiota homeostasis. In this study, we identified a nitric oxide synthase responsible for NO production in the shrimp Marsupenaeus japonicus. The expression of Nos and the NO concentration in the gastrointestinal tract were increased significantly in shrimp orally infected with Vibrio anguillarum. After RNA interference of Nos or treatment with an inhibitor of NOS, L-NMMA, NO production decreased and the gut bacterial load increased significantly in shrimp. Treatment with the NO donor, sodium nitroprusside, increased the NO level and reduced the bacterial load significantly in the shrimp gastrointestinal tract. Mechanistically, V. anguillarum infection increased NO level via upregulation of NOS and induced phosphorylation of ERK. The activated ERK phosphorylated the NF-κB-like transcription factor, dorsal, and caused nuclear translocation of dorsal to increase expression of antimicrobial peptides (AMPs) responsible for bacterial clearance. In summary, as a signaling molecule, NOS-produced NO regulates intestinal microbiota homeostasis by promoting AMP expression against infected pathogens via the ERK-dorsal pathway in shrimp. [ABSTRACT FROM AUTHOR]

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]

A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of <100 amino acids in eukaryotes (50 amino acids in prokaryotes). Studies have shown that several sORF-encoded peptides (SEPs) have important physiological functions in different organisms. Many ribosomal proteins belonging to SEPs play important roles in several cellular processes, such as DNA damage repair and apoptosis. Several studies have implicated SEPs in response to infection and innate immunity, but the mechanisms have been unclear for most of them. In this study, we identified a sORF-encoded ribosomal protein S27 (RPS27) in Marsupenaeus japonicus. The expression of MjRPS27 was significantly upregulated in shrimp infected with white spot syndrome virus (WSSV). After knockdown of MjRPS27 by RNA interference, WSSV replication increased significantly. Conversely, after Mj RPS27 overexpression, WSSV replication decreased in shrimp and the survival rate of the shrimp increased significantly. These results suggested that Mj RPS27 inhibited viral replication. Further study showed that, after Mj RPS27 knockdown, the mRNA expression level of Mj Dorsal, Mj Relish, and antimicrobial peptides (AMPs) decreased, and the nuclear translocation of Mj Dorsal and Mj Relish into the nucleus also decreased. These findings indicated that Mj RPS27 might activate the NF-κB pathway and regulate the expression of AMPs in shrimp after WSSV challenge, thereby inhibiting viral replication. We also found that Mj RPS27 interacted with WSSV's envelope proteins, including VP19, VP24, and VP28, suggesting that Mj RPS27 may inhibit WSSV proliferation by preventing virion assembly in shrimp. This study was the first to elucidate the function of the ribosomal protein Mj RPS27 in the antiviral immunity of shrimp. [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]

Galectins are a lectin family characterized by a conserved sequence motif in the carbohydrate recognition domain, which preferential binds to galactosyl moieties. However, few studies about the biological roles of galectins in invertebrates have been reported except for the galectin (CvGal1) from the eastern oyster Crassostrea virginica. Furthermore, galectins have been described in only a few crustacean species, and no functional studies have been reported so far. In this study, we identified and functionally characterized a galectin from the kuruma shrimp Marsupenaeus japonicus, which we designated MjGal. Upon Vibrio anguillarum challenge, expression of MjGal was up-regulated mostly in hemocytes and hepatopancreas, and the protein bound to both Gram-positive and Gram-negative bacteria through the recognition of lipoteichoic acid (LTA) or lipopolysaccharide (LPS), respectively. By also binding to the shrimp hemocyte surface, MjGal functions as an opsonin for microbial pathogens, promoting their phagocytosis. Further, as shown by RNA interference, MjGal participates in clearance of bacteria from circulation, and thereby contributes to the shrimp’s immune defense against infectious challenge. Elucidation of functional and mechanistic aspects of shrimp immunity will enable the development of novel strategies for intervention in infectious diseases currently affecting the shrimp farming industry worldwide. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a critical pathogenic bacterium for the kuruma shrimp, Marsupenaeus japonicus. Antibacterial drug application is the main method to control vibriosis in aquaculture; however, drug residues and the emergence of resistant bacteria have restricted their use. Therefore, the present study aimed to identify metabolites that are likely to play roles in the homeostasis of the intestinal microbiota, and to further explore their application in shrimp aquaculture. Metabolomic analysis, using high-performance liquid chromatography tandem mass spectrometry (HPLC/MS) of shrimp intestinal contents, was performed. The results showed that metabolic profiles changed distinctly in shrimp intestines after V. parahaemolyticus challenge compared with that in the control. The levels of 39 metabolites changed significantly after Vibrio challenge. Notably, vitamin D3 metabolism was enhanced in shrimp intestines after Vibrio challenge. Oral administration of 0.1 mg/kg vitamin D3 effectively improved the survival rate of shrimp infected with V. parahaemolyticus. Further studies indicated that vitamin D3 promoted bacterial clearance by regulating the expression of antimicrobial peptides genes in the shrimp intestines. Transcriptomics analysis revealed that vitamin D3 functioned in shrimp intestinal immunity by activating the prophenoloxidase system and Toll signaling pathway to induce the expression of certain antimicrobial peptides to maintain microbial homeostasis. Collectively, we identified vitamin D3 as an immunomodulatory factor that plays an important role in boosting the immune response against pathogenic bacteria in shrimp. • 39 metabolites changed significantly post V. parahaemolyticus challenge. • Vitamin D3 metabolism was enhanced in shrimp intestine post challenge. • Vitamin D3 improved shrimp survival rate. • Vitamin D3 promoted bacterial clearance by regulating the expression of AMPs. • Vitamin D3 functioned in shrimp intestinal immunity. [ABSTRACT FROM AUTHOR]

Galectins are a structurally conserved family of ß-galactoside-binding lectins characterized by a unique sequence motif in the carbohydrate recognition domain, and of wide taxonomic distribution, from fungi to mammals. Their biological functions, initially described as key to embryogenesis and early development via recognition of endogenous ("self") carbohydrate moieties, are currently understood as also encompassing tissue repair, cancer metastasis, angiogenesis, adipogenesis, and regulation of immune homeostasis. More recently, however, numerous studies have contributed to establish a new paradigm by revealing that galectins can also bind to exogenous ("non-self") glycans on the surface of potentially pathogenic virus, bacteria, and eukaryotic parasites, and function both as pathogen recognition receptors (PRRs) and effector factors in innate immunity. Our studies on a galectin from the kuruma shrimp Marsupenaeus japonicus (Mj Gal), revealed that it functions as a typical PRR. Expression of MjGal is upregulated by infectious challenge, and can recognize both Gram (+) and Gram (−) bacteria. MjGal also recognizes carbohydrates on the shrimp hemocyte surface, and can cross-link microbial pathogens to the hemocytes, promoting their phagocytosis and clearance from circulation. Therefore, MjGal contributes to the shrimp's immune defense against infectious challenge both as a PRR and effector factor. Our studies on galectins from the bivalve mollusks, however, have shown that although they can function in immune defense as MjGal, protistan parasites take advantage of the recognition roles of the host galectins, for successful attachment and host infection. We identified in the eastern oyster Crassostrea virginica two galectins (CvGal1 and CvGal2) that not only recognize a large variety of bacterial species, but also the protozoan parasite Perkinsus marinus. Like the shrimp MjGal, both oyster galectins function as opsonins, and promote parasite adhesion and phagocytosis. However, P. marinus survives intrahemocytic oxidative killing and proliferates, eventually causing systemic infection and death of the oyster host. In the softshell clam Mya arenaria we identified a galectin (MaGal1) that displays carbohydrate specificity and recognition properties for sympatric Perkinsus species (P. marinus and P. chesapeaki), that are different from CvGal1 and CvGal2. Our results suggest that although galectins from bivalves can function as PRRs, Perkinsus parasites have co-evolved with their hosts to subvert the galectins' immune functions for host infection by acquisition of carbohydrate-based mimicry. [ABSTRACT FROM AUTHOR]

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F 1 F 0 -ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (Mj ATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). Mj ATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant Mj ATPIF1 protein injection. Further study found that Mj ATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that Mj ATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation. • A mitochondrial ATPase inhibitor factor 1 was identified from kuruma shrimp. • Mj ATPIF1 was widely distributed in shrimp tissues and upregulated by WSSV challenge. • Mj ATPIF1 promoted the production of mitochondria superoxide. • Mj ATPIF1 triggered the activation of NF-κB by ROS. • Mj ATPIF1 benefits WSSV replication in kuruma shrimp. [ABSTRACT FROM AUTHOR]

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) typically composing of eight subunits (CSN1-8) mediates the process of deneddylation and deubiquitination. The fifth subunit of COP9 signalosome, CSN5, has special characteristics compared with the other seven subunits, and plays vital roles in the deneddylation activity and diverse cellular processes. However, the role of CSN5 in antiviral immunity is not clear. In this study, we identified 8 subunits (CSN1−8) of COP9 signalosome in shrimp Marsupenaeus japonicus. CSN1-6 were existed in all tested tissues, but CSN7-CSN8 were not detected in hepatopancreas. After WSSV challenged, the expression level of Csn1 to Csn4 , and Csn6 to Csn8 were highly decreased, but the expression level of Csn5 was conspicuously increased in shrimp challenged by white spot syndrome virus (WSSV). The CSN5 was recombinantly expressed in Escherichia coli and its polyclonal antibody was prepared. The expression level of CSN5 was conspicuously increased at RNA and protein levels in the shrimp challenged by WSSV. After knockdown of Csn5 by RNA interference, the WSSV replication was obviously increased in shrimp. When injected the recombinant protein of CSN5 with the membrane penetrating peptide into shrimp, WSSV replication was inhibited and the survival rate of shrimp was significantly improved compared with control. We further analyzed the expression of antimicrobial peptides (AMPs) in Csn5 -RNAi shrimp, and the results showed that the expression of several AMPs was declined significantly. These results indicate that CSN5 inhibits replication of WSSV via regulating expression of AMPs in shrimp, and the recombinant CSN5 might be used in shrimp aquaculture for the white spot syndrome disease control. • Eight subunits (CSN1−8) of COP9 signalosome are identified in Marsupenaeus japonicus. • Shrimp CSN subunits are quite similar in domain architecture with their counterparts from other species. • CSN5 is upregulated in shrimp challenged by WSSV. • CSN5 inhibits the replication of WSSV via enhancing expression of antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Viruses have developed superior strategies to escape host defenses or exploit host components and enable their infection. The forkhead box transcription factor O family proteins (FOXOs) are reportedly utilized by human cytomegalovirus during their reactivation in mammals, but if FOXOs are exploited by viruses during their infection remains unclear. In the present study, we found that the FOXO of kuruma shrimp (Marsupenaeus japonicus) was hijacked by white spot syndrome virus (WSSV) during infection. Mechanistically, the expression of leucine carboxyl methyl transferase 1 (LCMT1) was up-regulated during the early stages of WSSV infection, which activated the protein phosphatase 2A (PP2A) by methylation, leading to dephosphorylation of FOXO and translocation into the nucleus. The FOXO directly promoted transcription of the immediate early gene, wsv079 of WSSV, which functioned as a transcriptional activator to initiate the expression of viral early and late genes. Thus, WSSV utilized the host LCMT1-PP2A-FOXO axis to promote its replication during the early infection stage. We also found that, during the late stages of WSSV infection, the envelope protein of WSSV (VP26) promoted PP2A activity by directly binding to FOXO and the regulatory subunit of PP2A (B55), which further facilitated FOXO dephosphorylation and WSSV replication via the VP26-PP2A-FOXO axis in shrimp. Overall, this study reveals novel viral strategies by which WSSV hijacks host LCMT1-PP2A-FOXO or VP26-PP2A-FOXO axes to promote its propagation, and provides clinical targets for WSSV control in shrimp aquaculture. • WSSV upregulates expression of LCMT1 which is responsible for PP2A methylation. • WSSV exploits LCMT1-PP2A-FOXO axis to promote its replication during early infection. • WSSV utilizes VP26-PP2A-FOXO axis to promote viral replication during late infection. • FOXO targets Wsv079 to increase transcription of viral genes in WSSV infected shrimp. [ABSTRACT FROM AUTHOR]

Abstract Thymosins β are actin-binding proteins that play a variety of different functions in inflammatory responses, wound healing, cell migration, angiogenesis, and stem cell recruitment and differentiation. In crayfish, thymosins participate in antiviral immunology. However, the roles of thymosin during bacterial infection in shrimp remain unclear. In the present study, four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus , and named as Mj thymosin2, Mj thymosin3, Mj thymosin4, and Mj thymosin5 according the number of their thymosin beta actin-binding motifs. Mj thymosin3 was selected for further study because its expression level was the highest in hemocytes. Expression analysis showed that Mjthymosin3 was upregulated in hemocytes after challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant Mj thymosin3 protein could inhibit the growth of certain bacteria in an in vitro antibacterial test. Mj thymosins could facilitate external bacterial clearance in shrimp, and were beneficial to shrimp survival post V. anguillarum or S. aureus infection. The results suggested that Mj thymosins played important roles in the antibacterial immune response of kuruma shrimp. Highlights • Four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus. • Mjthymosin3 was upregulated in hemocytes after challenge by V. anguillarum or S. aureus. • R Mj thymosin3 could inhibit the growth of certain bacteria. • Mj thymosins could facilitate external bacterial clearance in shrimp. • Mj thymosins were beneficial to shrimp survival post V. anguillarum or S. aureus infection. [ABSTRACT FROM AUTHOR]

Abstract The cation-dependent mannose-6-phosphate receptor (CD-MPR) is a member of the P-type lectin family. As a type I transmembrane glycoprotein, it functions in the delivery of newly synthesized acid hydrolases from the trans-Golgi network to endosomes for their subsequent transfer to the lysosome by binding the mannose-6-phosphate receptor-recognition moieties in the hydrolases. However, the functions of CD-MPR in immune responses are seldom reported. In the present study, we identified a CD-MPR-like molecule in Marsupenaeus japonicus and designed it as Mj CD-MPR. It was significantly upregulated after challenge with Vibrio anguillarum at the mRNA and protein levels. Knockdown of MjCD-MPR resulted in a significant increase in the amount of V. anguillarum in the hemolymph of shrimp, which suggested that Mj CD-MPR plays a role in shrimp antibacterial defense. The recombinant extracytoplasmic region of Mj CD-MPR could bind gram-positive and gram-negative bacteria by interaction with peptidoglycan, lipopolysaccharide, and lipoteichoic acid. Mj CD-MPR showed no direct bacteriostatic or bacteriocidal activity. Knockdown of MjCD-MPR decreased the expression levels of several antimicrobial peptides ( Alf-C1, Alf-E1 , Crustin I-2 , and Crustin I-3 ), suggesting that Mj CD-MPR promotes the expression of antimicrobial peptides in shrimp. In summary, working as a pattern recognition receptor, Mj CD-MPR recognizes invading bacteria and triggers the expression of AMPs against bacterial infection in shrimp. Highlights • A CD-MPR identified in Marsupenaeus japonicus is upregulated in shrimp challenged by bacteria. • Mj CD-MPR promotes in vivo bacterial clearance and plays a role against bacterial infection in shrimp. • The extracytoplasmic region of Mj CD-MPR binds gram-positive and gram-negative bacteria by binding to polysaccharides. • Mj CD-MPR plays a role against bacterial infection by promoting the expression of antimicrobial peptides. [ABSTRACT FROM AUTHOR]

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

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]

Leucine rich repeat (LRR) motif exists in many immune receptors of animals and plants. Most LRR containing (LRRC) proteins are involved in protein-ligand and protein-protein interaction, but the exact functions of most LRRC proteins were not well-studied. In this study, an LRRC protein was identified from kuruma shrimp Marsupenaeus japonicus , and named as Mj LRRC1. MjLRRC 1 was consistently expressed in different tissues of normal shrimp with higher expression in gills and stomach. At the transcriptional level, there were no significant changes of MjLRRC 1 after injection of Vibrio anguillarum or Staphylococcus aureus in gills and hepatopancreas. While in V. anguillarum oral infection, MjLRRC 1 was upregulated in stomach but not in intestine. The recombinant Mj LRRC1 protein could bind to Gram-positive and Gram-negative bacteria, bacterial cell wall components including peptidoglycan, lipoteichoic acid, and lipopolysaccharide. Mj LRRC1 regulated the expression of some antimicrobial peptide (AMP) genes and participated in bacteria clearance of stomach. All these results suggested that Mj LRRC1 might play important roles in antibacterial immune response of kuruma shrimp. [ABSTRACT FROM AUTHOR]

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

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

The metazoan gut lumen harbors numerous microbial communities. Tolerance for high bacterial counts and maintenance of microbiota homeostasis remain insufficiently studied. In this study, we identified a novel dual oxidase ( MjDUOX2 ) involved in reactive oxygen species (ROS) production in the kuruma shrimp Marsupenaeus japonicus . Mj DUOX2 is a transmembrane protein with an N-signal peptide region (19 aa) and a peroxidase homology domain (PHD, 554 aa) in the extracellular region; seven transmembrane regions; and three EF (calcium-binding region) domains (110 aa), a FAD-binding domain (104 aa), and a NAD-binding domain (156 aa) in the intracellular region. The novel Mj DUOX2 exhibits a relatively low similarity (26.84% identity) to a previously reported DUOX in the shrimp (designated as Mj DUOX1). The mRNA of MjDUOXs was widely distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Oral infection of the shrimp with pathogenic bacteria upregulated the mRNA expression of Mj DUOXs and increased the ROS level in the intestine. However, High ROS level could inhibit the expression of MjDUOX s in shrimp after Vibrio anguillarum infection. Knockdown of MjDUOX s by RNA interference (RNAi) decreased the ROS level, increased the bacterial count in the intestine, and decreased the survival rate of the MjDUOX -RNAi shrimp infected with V. anguillarum . These results suggest that Mj DUOXs play an important role for microbiota homeostasis in intestine of shrimp. [ABSTRACT FROM AUTHOR]

Penaeidins are an important family of antimicrobial peptides (AMPs) in penaeid shrimp. To date, five groups of penaeidins have been identified in penaeid shrimp. All are composed of a proline-rich N-terminus and a C-terminus containing six cysteine residues engaged in three disulfide bridges. In this study, a new type of penaeidin from Marsupenaeus japonicus was identified. The full-length penaeidin contains a unique serine-rich region and a penaeidin domain, which consists of a proline-rich region and a cysteine-rich region. Here, we classify all penaeidins into two subfamilies. All reported penaeidins are in subfamily I, and the new penaeidin identified in M. japonicus is designated as Penaeidin subfamily II ( Mj Pen-II). Mj Pen-II was expressed in hemocytes, heart, hepatopancreas, gills, stomach and intestine, and was upregulated after bacterial challenge. A liquid bacteriostatic assay showed that Mj Pen-II had antibacterial activity to some Gram-positive and Gram-negative bacteria. Mj Pen-II could bind to bacteria by binding to polysaccharides on the surface of bacteria, thus promoting bacterial agglutination. The serine-rich region enhanced the agglutination activity of Mj Pen-II. The proline-rich domain had a stronger bacterial-binding activity and polysaccharide-binding activity than the cysteine-rich domain. Mj Pen-II was also found to be involved in the phagocytosis of bacteria and efficiently improved the phagocytosis rate. Therefore, Mj Pen-II eliminates bacteria through direct bacterial inhibition as well as by promoting phagocytosis in shrimp. [ABSTRACT FROM AUTHOR]

Scavenger receptors (SRs) are involved in innate immunity through recognizing pathogen-associated molecular patterns (PAMPs) and in pathogenesis of diseases through interactions with damage-associated molecular patterns (DAMPs). The roles of SRs in invertebrate innate immunity still need to be elucidated. Here we identify a class B scavenger receptor from kuruma shrimp, Marsupenaeus japonicus , designated Mj SR-B1. The recombinant Mj SR-B1 agglutinated bacteria in a calcium dependent manner and bound lipopolysaccharide and lipoteichoic acid. After knockdown of Mj SR-B1 , both the bacterial clearance and phagocytotic ability of M. japonicus against V. anguillarum and S. aureus were impaired, and several phagocytosis related genes were downregulated. The expression levels of antimicrobial peptides were also downregulated. Overexpression of Mj SR-B1 led to enhanced bacterial clearance, phagocytosis rate and upregulation of phagocytosis-related and antimicrobial peptide genes. However, overexpression of mutant Mj SR-B1ΔC, which lacks the carboxyl tail of Mj SR-B1, had none of these effects. Our results indicate that Mj SR-B1 can protect shrimp from bacteria by promoting phagocytosis and by enhancing the expression of antimicrobial peptides. [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]

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]

Highlights: [•] A serine protease inhibitor (MjSerp1) was identified in kumura shrimp, Marsupenaeus japonicus. [•] MjSerp1 is upregulated in the hepatopancreas after Vibrio anguillarum challenge. [•] MjSerp1 closely binds to bacteria and inhibits activity of their proteases. [•] MjSerp1 exhibits antibacterial activity. [•] MjSerp1 enhances bacterial clearance in vivo. [Copyright &y& Elsevier]

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

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

Dyslexia-associated protein KIAA0319, with immunoglobulin-like polycystic kidney disease (PKD) domains, is a dyslexia susceptibility protein. KIAA0319 participates in neuronal migration and is involved in the interaction between neurons and glial fibers. Recent studies found that KIAA0319 acts as a viral receptor via an interaction between its PKD domains with viruses. KIAA0319 mediates viral internalization via a clathrin-dependent pathway involving interaction with adaptor protein 2 (AP2). In the present study, we identified a cDNA encoding a dyslexia-associated KIAA0319-like protein from Marsupenaeus japonicus (designated as Mj KIAA0319L). MjKIAA0319L was upregulated in shrimp challenged by white spot syndrome virus (WSSV). After knockdown of MjKIAA0319L using RNA interference, followed by WSSV infection in shrimp, WSSV replication increased and the shrimp survival rate decreased significantly. Further study found that the extracellular domain of Mj KIAA0319L expressed in Escherichia coli bound WSSV particles by interacting with the WSSV envelope protein VP28. Fluorescent immunocytochemical analysis showed that Mj KIAA0319L was internalized into the cytoplasm of hemocytes during WSSV infection. The intracellular domain of Mj KIAA0319L, comprising a YXXΦ motif, interacted with AP2μ, suggesting that Mj KIAA0319L binds to WSSV and induces endocytosis in a clathrin-dependent manner. Therefore, Mj KIAA0319L recognizes WSSV via its extracellular domain and induces endocytosis of WSSV through its intracellular domain in shrimp. Thus, the Mj KIAA0319L-AP2-clathrin pathway is involved in the phagocytosis of WSSV to restrict viral infection in shrimp. • KIAA0319-like mRNA expression was upregulated in kuruma shrimp challenged by WSSV. • KIAA0319L binds to WSSV via an interaction with viral envelope protein VP28. • The intracellular region of KIAA0319L interacts with AP2 and induces endocytosis. • The KIAA0319L-AP2-clathrin pathway is involved in the phagocytosis of WSSV. [ABSTRACT FROM AUTHOR]